Fatty alcohol oxidase genes and proteins from Candida tropicalis and methods relating thereto

ABSTRACT

The present invention provides fatty alcohol oxidase (FAO) proteins and nucleic acid molecules encoding the FAO proteins. Also provided are analogs, derivatives, and enzymatically active fragments of the FAO proteins. Vectors and host cells comprising the nucleic acid molecules encoding the FAO proteins, analogs, derivatives and enzymatically active fragments thereof are also provided. In addition, FAO signature peptides and isolated nucleic acid molecules encoding the signature peptides are provided by the present invention. Methods of producing or increasing production of a subject FAO protein, methods for increasing aldehyde production during the second step of the ω-oxidation pathway of fatty acids, methods for increasing production of a ketone from an alcohol during the second step of the ω-oxidation pathway of fatty acids, and methods for increasing production of a dicarboxylic acid are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims benefit of U.S. Provisional ApplicationSer. No. 60/374,021, filed Apr. 19, 2002, which application isincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded, at least in part, under a grant from theDepartment of Commerce, NIST-ATP Cooperative Agreement Number70NANB8H4033. The Government may therefore have certain fights in theinvention.

BACKGROUND OF THE INVENTION

Several genetically engineered strains of Candida tropicalis have beenused in fermentations for the bioconversion of the 18-carbon fatty acidknown as oleic acid to 18-carbon dicarboxylic acid. When grown on fattyacids, wild-type C. tropicalis converts long chain fatty acids to acetylCoA by a process known as β-oxidation, which is the sequentialcatabolism of 2-carbon length fragments of a fatty acid to acetyl CoA.β-Oxidation is thus named because the initial oxidative attack occurs atthe second carbon atom from the carboxylic group. C. tropicalis can alsocatabolize fatty acids through an ω-oxidation pathway in which only theterminal methyl carbon is oxidized to a carboxylic acid, yielding adicarboxylic acid. In ω-oxidation, the fatty acid is converted todicarboxylic acid along a three-step pathway beginning with theoxidation of the terminal methyl group to an alcohol. This step iscatalyzed by the hydroxylase complex that contains both the cytochromeP450 monooxygenase (CYP) and cytochrome P450 reductase (NCP) proteins.The alcohol is then converted to an aldehyde by fatty alcohol oxidase(FAO) and then to the dicarboxylic acid by an aldehyde dehydrogenase.The desired product is the long-chain dicarboxylic acid. A fatty alcoholoxidase is distinguished from an alcohol oxidase in its chain lengthspecificity. Alcohol oxidases in general are specific for methanol butcan sometimes oxidize alcohols up to C4. Fatty alcohol oxidasesgenerally do not oxidize alcohols with chain lengths less than eight.

In wild-type Candida tropicalis, β-oxidation consumes fatty acids muchfaster than the ω-oxidation pathway can oxidize them. However, byinactivating the POX 4 and POX 5 genes, which gene products areresponsible for the initiation of β-oxidation, such geneticallyengineered C. tropicalis strains preferentially shunt fatty acids intothe ω-oxidation pathway. The base strain used for the development ofvarious gene-amplified strains is H5343, which is C. tropicalis strain20336 (American Type Culture Collection) with both POX 4 and POX 5 genesinactivated by insertional inactivation. The primary strain used inlarger-scale production fermentations is HDC23-3, which is derived fromH5343, but also has the CYP52A2 (a cytochrome P450 monooxygenase) geneamplified. The hydroxylase complex is responsible for catalyzing thefirst step in ω-oxidation, which is considered the rate-limiting step.Amplification of the CYP52A2 and NCP genes help to overcome thisrate-limitation, but then the next bottleneck becomes the conversion ofthe alcohol to the aldehyde by the FAO enzyme. During fermentations withHDC23-3, it has been discovered that a small amount (ca. 0.5% w/w inbroth) of ω-hydroxy fatty acid (HFA) accumulates during thefermentation. This partial oxidation product interferes with laterpurification steps and causes lower overall yields. There is anadditional need therefore, for reducing the bottleneck in the conversionof the alcohol to an aldehyde during the second step of the ω-oxidationpathway.

A small number of fatty alcohol oxidases have been described in thescientific literature in various yeasts, examples of which are Candidatropicalis (1, 2, 3, 4), Candida maltosa (5,6), Candida cloacae (4),Torulopsis candida (7), Candida (Torulopis) bombicola (8), and Candida(Torulopsis) apicola (9). The FAO was purified from the hexadecane-grownyeast, T. candida (7) and described as a tetramer (mw 290 kD) withsubunit mol. wt. of 75 kD. It has a pH optimum of 7.6 and oxidizeshigher alcohols with a carbon chain length of C4 to C16.Hexadecane-grown C. bombicola (8) apparently has two different alcoholoxidase activities, one with an optimal chain length specificity of 10for n-alcohols and another with an optimal chain length specificity of14. The FAO from C. maltosa (6) catalyzes the oxidation of 1-alkanols(C4 to C22) with highest activity utilizing 1-octanol. It also oxidizes2-alkanols (C8 to C16). α,ω-Alkanediols, ω-hydroxypalmitic acid,phenylalkanols and terpene alcohols were all found to be substrates forthe FAO, but at fairly low rates of oxidation. The oxidation of2-alkanols is stereoselective for the R(−) enantiomers only.

The FAO from C. tropicalis (ATCC 20336) grown on hexadecane was firstdescribed by Kemp et al. in 1988 (1). It was found to oxidize 1-alkanolsfrom C4 to C18, but has a maximal activity with dodecanol. It was foundto oxidize 16-hydroxypalmitate but not 12-hydroxylaurate. The FAO waslater purified (3) and was shown to be a dimer (mw=145 kD) with subunitmolecular weight of 68–72 kD. The purified enzyme showed similarsubstrate specificity as described previously, but demonstratedadditional activity with 12-hydroxylaurate and 2-dodecanol. The enzymewas found to be a light sensitive flavoprotein, but the identity of theflavin was not known.(10).

Recently two FAO genes from C. cloacae and one FAO gene from C.tropicales were cloned and the DNA sequences determined (4, 12). Theopen reading frame (ORF) for FAO1 and FAO2 from C. cloacae were 2094 bpand 2091 bp, respectively. The ORF for FAOT from C. tropicalis was 2112bp. FAOT shares 60.6% and 61.7% nucleotide identities and 74.8% and76.2% amino acid sequence similarities with C. cloacae FAO1 and FAO2,respectively. The FAO1 gene but not the FAO2 gene has been successfullycloned and expressed in Escherichia coli.

The present invention provides FAO genes from C. tropicalis andcompositions and methods employing the FAO genes. The compositions andmethods are useful for increasing FAO activity during the second step ofomega-oxidation of fatty acids and ultimately result in an increase indiacid productivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of FAO1jf which is the coding region of theFAO1 gene ligated into the expression vector pJF118EH.

FIG. 2 is a restriction map of FAO2jf which is the coding region of theFAO2 gene ligated into the expression vector pJF118EH.

FIG. 3 graphically depicts typical alcohol oxidase activity in lab-scalefermentations with oleic acid as substrate.

FIG. 4 graphically depicts activity of FAO1, FAO2a, and FAO2a′ on1-alkanols.

FIG. 5 graphically depicts activity of FAO1, FAO2a and FAO2a′ on2-alkanols.

FIG. 6 graphically depicts activity of FAO1, FAO2, and FAO2a′ on otheralkanols.

FIG. 7 graphically depicts a comparison of productivity in fermentationswith FAO-amplified strains or base strain, H5343.

FIG. 8 graphically depicts a comparison of ω-hydroxyfatty acidconcentration in fermentations with FAO-amplified strain, HDC40-7, orbase strain, H5343.

FIG. 9 graphically depicts a comparison of alcohol oxidase activitybetween the FAO1-amplified strain, HDC40-7, and the base strain, H5343.

FIG. 10 graphically depicts a comparison of ω-hydroxyfatty acidconcentration in fermentations with FAO-amplified strains, HDC40-1,HDC40-5, or HDC40-7, using ricinoleic acid as substrate.

SUMMARY OF THE INVENTION

The present invention provides fatty alcohol oxidase proteins comprisingthe amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 10, or 12,including analogs, derivatives, or enzymatically active fragmentsthereof. Also provided are isolated nucleic acid molecules encodingfatty alcohol oxidase proteins comprising the amino acid sequences setforth in SEQ ID NOs: 2, 4, 6, 10, or 12, including analogs, derivatives,or enzymatically active fragments thereof. Examples of such isolatednucleic acid molecules include those having the sequences set forth inSEQ ID NOs: 1, 3, 5, 9, and 11.

The present invention also provides a peptide consisting of orcomprising the sequence set forth in any one of SEQ ID NO:13, SEQ IDNO:14, or SEQ ID NO:19.

In addition, the present invention also provides a fatty alcohol oxidaseor enzymatically active fragment thereof having an amino acid sequenceidentity of at least one of: greater than 82% when compared to the aminoacid sequence set forth in SEQ ID NO:2, greater than 83% when comparedto the amino acid sequence set forth in SEQ ID NO:2, greater than 84%when compared to the amino acid sequence set forth in SEQ ID NO:2,greater than 85% when compared to the amino acid sequence set forth inSEQ ID NO:2, greater than 90% when compared to the amino acid sequenceset forth in SEQ ID NO:2, or greater than 95% when compared to the aminoacid sequence set forth in SEQ ID NO:2. Preferably, this fatty alcoholoxidase further comprising a signature peptide having the amino acidsequence set forth in SEQ ID NO:13.

A fatty alcohol oxidase or enzymatically active fragment thereof havingan amino acid sequence identity of at least one of: greater than 85%when compared to the amino acid sequence set forth in SEQ ID NO:4,greater than 86% when compared to the amino acid sequence set forth inSEQ ID NO:4, greater than 87% when compared to the amino acid sequenceset forth in SEQ ID NO:4, greater than 88% when compared to the aminoacid sequence set forth in SEQ ID NO:4 greater than 90% when compared tothe amino acid sequence set forth in SEQ ID NO:4, or greater than 95%when compared to the amino acid sequence set forth in SEQ ID NO:4 isalso provided. Preferably, this fatty alcohol oxidase comprises asignature peptide having the amino acid sequence set forth in at leastone of SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:19.

Also in accordance with the present invention, there is provided a fattyalcohol oxidase or enzymatically active fragment thereof having an aminoacid sequence identity of at least one of: greater than 85% whencompared to the amino acid sequence set forth in SEQ ID NO:6, greaterthan 86% when compared to the amino acid sequence set forth in SEQ IDNO:6, greater than 87% when compared to the amino acid sequence setforth in SEQ ID NO:6, greater than 88% when compared to the amino acidsequence set forth in SEQ ID NO:6 greater than 90% when compared to theamino acid sequence set forth in SEQ ID NO:6, or greater than 95% whencompared to the amino acid sequence set forth in SEQ ID NO:6.Preferably, such a fatty alcohol oxidase also comprises a signaturepeptide having the sequence set forth in at least one of SEQ ID NO:13,SEQ ID NO:14 or SEQ ID NO:19.

In addition, there is provided an isolated nucleic acid moleculeencoding the signature motif set forth in SEQ ID NO:13. For example, theisolated nucleic acid may comprise the nucleotide sequence:

TGY GGN TTY TGY TAY YTN GGN TGY

as set forth in SEQ ID NO: 32, wherein:

Y is C or T, and N is A or G, or C or T.

Also provided is an isolated nucleic acid molecule encoding thesignature motif set forth in SEQ ID NO:14. For example, such an isolatednucleic acid molecule may comprise the nucleotide sequence: ATH ATH GGNWSN GGN GCN GGN GCN GGN GTN AUG GCN wherein:

R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is Aor T, H is A or T or C, B is G or T or C, D is G or A or T, N is A or G,or C or T.

The present invention also provides an isolated nucleic acid moleculeencoding the signature motif set forth in SEQ ID NO:19. An example ofsuch a nucleotide sequence includes an isolated nucleic acid moleculecomprising the nucleotide sequence:

GCN GGN WSN ACN YTN GGN GGN GGN

wherein R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C,W is A or T, H is A or T or C, B is G or T or C, D is G or A or T, N isA or G, or C or T.

The present invention also provides an isolated nucleic acid moleculeencoding the signature motif set forth in SEQ ID NO:13 and having asequence identity of greater than 77% compared to the nucleotidesequence set forth in SEQ ID NO:1; an isolated nucleic acid moleculeencoding the signature motif set forth in SEQ ID NO:14 and/or SEQ IDNO:13 and/or SEQ ID NO:19 and having a sequence identity of greater than78% compared to the nucleotide sequence set forth in SEQ ID NO:3; anisolated nucleic acid molecule encoding the signature motif set forth inSEQ ID NO:14 and/or SEQ ID NO:13 and/or SEQ ID NO:19 and having asequence identity of greater than 79% compared to the nucleotidesequence set forth in SEQ ID NO:5.

In addition, the present invention also provides an isolated nucleicacid molecule comprising a nucleotide sequence which encodes thesignature motif set forth in SEQ ID NO:13 and which hybridizes undermedium to high stringency conditions to nucleotides 1941–4054 of thenucleotide sequence set forth in SEQ ID NO:1, and an isolated nucleicacid molecule comprising a nucleotide sequence which encodes thesignature motif set forth in SEQ ID NO:13 and/or SEQ ID NO:14 and/or SEQID NO:19 and which hybridizes under medium to high stringency conditionsto nucleotides 1521–3635 of the nucleotide sequence set forth in SEQ IDNO:3 or nucleotides 1094–3213 of the nucleotides set forth in SEQ IDNO:5.

In accordance with the present invention, there is also provided anisolated nucleic acid molecule comprising an open reading frame (ORF)for a fatty alcohol oxidase (FAO) gene from Candida tropicalis whereinthe ORF is operably linked to a promoter which is capable of affectingexpression of the ORF. Preferably, the FAO comprises the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:10, or SEQ ID NO:12. In another preferred embodiment, the ORFcomprises nucleotides 1941–4054 of the nucleotide sequence set forth inSEQ ID NO:1, nucleotides 1521–3635 of the nucleotide sequence set forthin SEQ ID NO:3, or nucleotides 1099–3213 of the nucleotide sequence setforth in SEQ ID NO:5.

Vectors comprising the isolated nucleic acid molecules described hereinare also provided. Such vectors include but are not limited to plasmids,phagemids, phage, cosmids, or linear DNA vectors. In addition, hostcells are also provided which host cells comprise a subject vector orisolated nucleic acid molecule. Examples of host cells include bacterialcells, fungal cells, insect cells, animal cells or plant cells. examplesof fungal cells include those of Yarrowia sp., Bebaromyces sp.,Saccharomyces sp., Schizosaccharomyces sp., Pichia sp. and Candida sp.Host cells of Candida tropicalis are especially preferred.

The present invention also provides a method of producing an FAO1protein. The method comprises: transforming a suitable host cell with aDNA sequence encoding a protein having the amino acid sequence as setforth in SEQ ID NO:2; and culturing the cell under conditions favoringexpression of the FAO1 protein.

In addition, the present invention provides a method of producing anFAO2a protein. The method comprises the steps of: transforming asuitable host cell with a DNA sequence that encodes a protein having theamino acid sequence as set forth in SEQ ID NO:4; and culturing the cellunder conditions favoring expression of the FAO2a protein.

A method of producing an FAO2b protein is further provided. The methodcomprises the steps of transforming a suitable host cell with a DNAsequence that encodes a protein having the amino acid sequence as setforth in SEQ ID NO:6; and culturing the cell under conditions favoringthe expression of the protein.

In accordance with the present invention, there is also provided amethod for increasing production of an aldehyde during the second stepof the ω-oxidation pathway of fatty acids. The method comprises thesteps of:

(a) providing a host cell having a naturally occurring number of FAOgenes;

(b) increasing, in the host cell, the number of FAO1 genes which encodean FAO1 protein having the amino acid sequence as set forth in SEQ IDNO:2; and

(c) culturing the host cell in media containing an organic substratewhich upregulates the FAO1 gene, to effect increased production of analdehyde. Preferably, the FAO1 gene comprises nucleotides 1941–4054 ofthe nucleotide sequence as set forth in SEQ ID NO:1.

Also provided by the present invention is a method for increasingproduction of an aldehyde during the second step of the ω-oxidationpathway of fatty acid. The method comprises the steps of:

(a) providing a host cell having a naturally occurring number of FAOgenes;

(b) increasing, in the host cell, the number of FAO2 genes which encodean FAO2a protein having the amino acid sequence as set forth in SEQ IDNO:4; and

(c) culturing the host cell in media containing an organic substratewhich upregulates the FAO2 gene, to effect increased production of analdehyde. Preferably, the FAO2a gene comprises nucleotides 1521–3635 ofthe nucleotide sequence as set forth in SEQ ID NO:3.

A method for increasing production of an aldehyde during the second stepof the ω-oxidation pathway of fatty acids is still further provided. Themethod comprises the steps of:

(a) providing a host cell having a naturally occurring number of FAOgenes;

(b) increasing, in the host cell, the number of FAO2 genes which encodean FAO2b protein having the amino acid sequence as set forth in SEQ IDNO:6; and

(c) culturing the host cell in media containing an organic substratewhich upregulates the FAO2b gene, to effect increased production of analdehyde. Preferably, the FAO2b gene comprises nucleotides 1099–3213 ofthe nucleotide sequence as set forth in SEQ ID NO:5.

A method for increasing production of a dicarboxylic acid is alsoprovided. The method comprises: (a) providing a host cell having anaturally occurring number of FAO genes; (b) increasing, in the hostcell, the number of FAO1 genes which encode an FAO1 protein having theamino acid sequence as set forth in SEQ ID NO:2; and (c) culturing thehost cell in media containing an organic substrate which upregulates theFAO1 gene, to effect increased production of dicarboxylic acid.Preferably, the FAO1 gene comprises nucleotides 1941–4054 of thenucleotide sequence as set forth in SEQ ID NO:1.

The present invention still further provides a method for increasingproduction of a dicarboxylic acid, said method comprising: (a) providinga host cell having a naturally occurring number of FAO genes; (b)increasing, in the host cell, the number of FAO2a genes which encode anFAO2 protein having the amino acid sequence as set forth in SEQ ID NO:4;and (c) culturing the host cell in media containing an organic substratewhich upregulates the FAO2a gene, to effect increased production ofdicarboxylic acid. Preferably, the FAO2a gene comprises nucleotides1521–3635 of the nucleotide sequence as set forth in SEQ ID NO:3.

In still another embodiment of the present invention, there is provideda method for increasing production of a dicarboxylic acid. The methodcomprises the steps of: (a) providing a host cell having a naturallyoccurring number of FAO genes; (b) increasing, in the host cell, thenumber of FAO2b genes which encode an FAO2 protein having the amino acidsequence as set forth in SEQ ID NO:6; and (c) culturing the host cell inmedia containing an organic substrate which upregulates the FAO2b gene,to effect increased production of dicarboxylic acid. Preferably, theFAO2b gene comprises nucleotides 1099–3213 of the nucleotide sequence asset forth in SEQ ID NO:5.

In still another embodiment, the present invention provides a method forincreasing the production of an FAO1 protein having an amino acidsequence as set forth in SEQ ID NO:2. The method comprises the steps of:

(a) transforming a host cell having a naturally occurring level of FAO1protein with an increased copy number of an FAO1 gene that encodes theFAO1 protein having the amino acid sequence as set forth in SEQ ID NO:2;and

(b) culturing the cell and thereby increasing expression of the proteincompared with that of a host cell containing a naturally occurring copynumber of the FAO1 gene.

A method for increasing the production of an FAO2 protein having anamino acid sequence as set forth in SEQ ID NO:4 is further provided. Themethod comprises:

(a) transforming a host cell having a naturally occurring amount of FAO2protein with an increased copy number of an FAO2 gene that encodes theFAO2 protein having the amino acid sequence as set forth in SEQ ID NO:4;and

(b) culturing the cell and thereby increasing expression of the proteincompared with that of a host cell containing a naturally occurring copynumber of the FAO2 gene.

In yet another aspect of the invention, there is provided a method forincreasing production of an aldehyde during the second step of theω-oxidation pathway of fatty acids. The method comprises the steps of:

(a) isolating a nucleic acid molecule comprising coding sequence for anFAO1 gene;

(b) isolating a promoter sequence from a gene which transcribes at ahigher rate than the FAO1 gene;

(c) operably linking the promoter sequence to the open reading frame(ORF) of the FAO1 gene to create a fusion gene;

-   -   (d) inserting the fusion gene into an expression vector;

(e) transforming a host cell with the expression vector and (f)culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the nucleic acid molecule encodes an FAO1 comprising theamino acid sequence set forth in SEQ ID NO:2.

A method for increasing production of an aldehyde from an alcohol duringthe second step of the ω-oxidation pathway of fatty acids is stillfurther provided. The method comprises the steps of:

(a) isolating a nucleic acid molecule comprising coding sequence for anFAO2 gene;

(b) isolating a promoter sequence from a gene which transcribes at ahigher rate than the FAO2 gene,

(c) operably linking the promoter sequence to the open reading frame(ORF) of the FAO2 gene to create a fusion gene;

(d) inserting the fusion gene into an expression vector;

(e) transforming a host cell with the expression vector and

(f) culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the nucleic acid molecule encodes an FAO2 protein comprisingthe amino acid sequence as set forth in SEQ ID NOs:4 or 6.

A method of increasing production of an ketone from an alcohol duringthe second step of the ω-oxidation pathway of fatty acids isadditionally provided. The method comprises the steps of:

(a) isolating a nucleic acid molecule comprising coding sequence for anFAO2 gene;

b) isolating a promoter sequence from a gene which transcribes at ahigher rate than the FAO2 gene,

(c) operably linking the promoter sequence to the open reading frame(ORF) of the FAO2 gene to create a fusion gene;

(d) inserting the fusion gene into an expression vector;

(e) transforming a host cell with the expression vector and

(f) culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the nucleic acid molecule encodes an FAO2 protein comprisingthe amino acid sequence as set forth in SEQ ID NOs:4 or 6.

In still another aspect of the invention, there is provided a method forincreasing dicarboxylic acid production. The method comprises the stepsof:

(a) isolating a nucleic acid molecule comprising coding sequence for anFAO1 gene;

(b) isolating a promoter sequence from a gene which transcribes at ahigher rate than the FAO1 gene;

(c) operably linking the promoter sequence to the open reading frame(ORF) of the FAO1 gene to create a fusion gene;

(d) inserting the fusion gene into an expression vector;

(e) transforming a host cell with the expression vector; and

(f) culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the ORF of the FAO1 gene comprises nucleotides 1941–4054 ofthe nucleotide sequence set forth in SEQ ID NO:1.

A method of increasing dicarboxylic acid production is still furtherprovided. The method comprises the steps of:

(a) isolating a nucleic acid molecule comprising coding sequence for anFAO2 gene;

(b) isolating a promoter sequence from a gene which transcribes at ahigher rate than the FAO2 gene,

(c) operably linking the promoter sequence to the open reading frame(ORF) of the FAO2 gene to create a fusion gene;

(d) inserting the fusion gene into an expression vector; (d)transforming a host cell with the expression vector; and

(e) culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the ORF of the FAO2 gene comprises nucleotides 1521–3635 ofthe nucleotide sequence as set forth in SEQ ID NO:3 or nucleotides1099–3213 of the nucleotide sequence set forth in SEQ ID NO:5.

DETAILED DESCRIPTION OF THE INVENTION

Diacid productivity is improved according to the present invention byselectively increasing enzymes which are known to be important to theoxidation of organic substrates such as fatty acids or aliphaticcompounds, such as alkanes. In accordance with the present invention,two fatty alcohol oxidase (FAO) genes of C. tropicalis, have beenidentified and characterized. The FAO genes encode fatty alcohol oxidaseenzymes, which catalyze the conversion of an alcohol to an aldehydeduring the second step of the ω-oxidation pathway. Amplification of theFAO gene copy number and/or transcriptional activity in a host cellresults in higher alcohol oxidase activity, higher diacid productivity,and, when fatty acid substrates are used, lower ω-hydroxy fatty acidlevels.

The present invention provides nucleotide sequences from C. tropicalisthat encode two different fatty alcohol oxidases, each of which has adifferent substrate specificity. In one embodiment, there is provided anisolated nucleic acid molecule which encodes the fatty alcohol oxidaseenzyme FAO1, having the amino acid sequence as set forth in SEQ ID NO:2.An example of such an isolated nucleic acid molecule includes the FAO1gene having the nucleotide sequence as set forth in SEQ ID NO:1.

In another aspect of the invention, there are provided isolated nucleicacid molecules that encode the fatty alcohol oxidase enzyme FAO2. Inaccordance with the present invention, two alleles have been identifiedand isolated which encode the FAO2 enzyme. The two alleles, FAO2a andFAO2b, are 95% identical by DNA sequence and have 98% similarity byamino acid sequence. The FAO2a enzyme comprises the amino acid sequenceas set forth in SEQ ID NO:4. An example of a nucleotide sequence whichencodes the FAO2a enzyme is set forth in SEQ ID NO:3. The FAO2b enzymecomprises the amino acid sequence as set forth in SEQ ID NO:6. Anexample of a nucleotide sequence which encodes the FAO2b enzyme is setforth in SEQ ID NO:5.

It has recently been determined that certain eukaryotes, e.g., certainyeasts, do not adhere, in some respects, to the “universal” genetic codewhich provides that particular codons (triplets of nucleic acids) codefor specific amino acids. Indeed, the genetic code is “universal”because it is virtually the same in all living organisms. CertainCandida sp. are now known to translate the CTG codon (which, accordingto the “universal” code designates leucine), as serine. See, e.g., Uedaet al., Biochemie (1994) 76, 1217–1222, where C. tropicalis, C.cylindracea, C. guilliermodii and C. lusitaniae are shown to adhere tothe “non-universal” code with respect to the CTG codon. Accordingly,nucleic acid sequences may code for one amino acid sequence in“universal” code organisms and a variant of that amino acid sequence in“non-universal” code organisms depending on the presence of CTG codonsin the nucleic acid coding sequence. The difference may become evidentwhen, in the course of genetic engineering, a nucleic acid moleculeencoding a protein is transferred from a “non-universal” code organismto a “universal” code organism or vice versa. Obviously, there will be adifferent amino acid sequence depending on which organism is used toexpress the protein.

Thus, the present invention also provides an amino acid sequence (setforth in SEQ ID NO:10) for an FAO2a enzyme when FAO2a is expressed in aspecies of Candida such as C. tropicalis. The amino acid sequence setforth in SEQ ID NO:10 has a serine residue at position 177. An exampleof a nucleotide sequence encoding the amino acid sequence set forth inSEQ ID NO:10 is provided by the nucleotide sequence set forth in SEQ IDNO:9 where a TCG codon is substituted for a CTG codon at position2049–2051.

The present invention also provides an amino acid sequence (set forth inSEQ ID NO:12) for an FAO2b enzyme when FAO2b is expressed in a speciesof Candida such as C. tropicalis. The amino acid sequence set forth inSEQ ID NO:12 has a serine residue at position 177. An example of anucleotide sequence encoding the amino acid sequence set forth in SEQ IDNO:12 is provided by the nucleotide sequence set forth in SEQ ID NO:11where a TCG codon is substituted for a CTG codon at positions 1627–1629.

The present invention also provides FAO proteins. For example, there isprovided an FAO1 protein having the amino acid sequence as set forth inSEQ ID NO:2 or an enzymatically active fragment thereof. Further, thereis provided an FAO2a protein comprising the amino acid sequence as setforth in SEQ ID NO:4 or an enzymatically active fragment thereof. Inanother embodiment, there is provided an FAO2b protein comprising theamino acid sequence as set forth in SEQ ID NO:6 or an enzymaticallyactive fragment thereof. In yet another embodiment, there is provide anFAO2a protein comprising the amino acid sequence as set forth in SEQ IDNO:10 or an enzymatically active fragment thereof. In still anotherembodiment, an FAO2b protein having the amino acid sequence as set forthin SEQ ID NO:12 or an enzymatically active fragment thereof is provided.As used herein, “enzymatically active fragment” refers to a portion ofthe FAO enzyme which is sufficient to retain enzymatic activity inconverting an alcohol to an aldehyde.

In accordance with the present invention, FAO1 and FAO2 proteins may beprepared by methods familiar to those skilled in the art such as bycloning the FAO1 and FAO2 genes (including the FAO2a and/or FAO2balleles) into an appropriate expression vector followed by expression ina suitable host cell. See, e.g., U.S. Pat. No. 6,331,420, incorporatedby reference herein as if fully set forth. The relevant enzyme orfragment thereof, may also be generated by direct amplification ofcorresponding coding sequence via PCR, followed by standard recombinantprocedures and expression in a suitable host cell. With respect to FAOanalogs, derivatives, FAO-like molecules, portions or enzymaticallyactive fragments thereof (as defined herein), PCR primers may bedesigned to allow direct amplification of coding sequences forcorresponding amino acid insertional, deletional, or substitutionalamino acid variants (as defined herein). Primers for use in PCR may besynthetic oligonucleotides prepared on an automated oligonucleotidesynthesizer such as an ABI DNA synthesizer available from Perkin-ElmerCorporation. In addition, oligonucleotides may be purchased fromcommercial manufacturers, for example, from Synthetic Genetics (SanDiego, Calif.). FAO1 and FAO2 proteins may also be chemicallysynthesized using well-known methodologies.

The appropriate DNA sequence may be inserted into a vector by a varietyof procedures deemed to be within the scope of those skilled in the art,which can include insertion of the DNA into an appropriate restrictionendonuclease site(s) or cloning the DNA sequence into the expressionvectors using high fidelity polymerase chain reaction. Standardtechniques for the construction of such vectors are well-known to thoseof ordinary skill in the art and may be found in references such asSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., or any of themyriad of laboratory manuals on recombinant DNA technology which arewidely available. A variety of strategies are available for ligatingfragments of DNA, the choice of which depends on the nature of thetermini of the DNA fragments. The vector may also include appropriatesequences for amplifying expression, or sequences that facilitatecloning, expression or processing.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or tetracycline or ampicillinresistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the enzyme. “Transformation” includes all forms of causinguptake of foreign DNA by a host cell. The transformed cells are thenscreened for those that contain the desired DNA and the successfultransformants are cultured under conditions that affect the expressionof the coding sequences.

Representative examples of appropriate hosts include bacterial cells,such as E. coli and Streptomyces; fungal cells, such as yeast; insectcells such as Drosophila S2 and Spodoptera Sf9; animal cells;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein and can include expression host BL21 or BL21 CodonPlusRIL strain (Stratagene, La Jolla, Calif.).

Following transformation of the host strain, the enzyme is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. If the FAO coding sequenceis under control of its native promoter, oleic acid may be used toinduce expression. If the FAO is operably linked to a heterologouspromoter, other inducers may be used. One preferred method of inductionis through the use of IPTG (Isopropyl-β-D-thiogalactopyranoside).

Cells are typically disrupted by physical or chemical means known tothose skilled in the art, centrifuged, and the resulting crude extractretained for further purification. Purification of FAO1 or FAO2 orfragment thereof can be carried out by any means known to those skilledin the art and can include chromatographic techniques, such ashistidine-tag affinity chromatography and Nickel affinitychromatography. Commercially available kits for this purification can bepurchased from vendors (Qiagen, Inc. Chatsworth, Calif.; Novagen,Calif., USA). An immunoassay, such as a Western blot assay, can then beutilized to verify the presence of the recombinant enzyme.

Also in accordance with the present invention, three different signaturepeptide sequences which are unique to the FAO1 and FAO2 proteins of thepresent invention have been discovered. The first signature peptide hasthe amino acid sequence: CGFCYLGC (SEQ ID NO:13). This first signaturepeptide is found in both FAO1 and FAO2 proteins of the present inventionbut not in other previously characterized yeast FAO proteins. Withreference to SEQ ID NO:2 (FAO1 protein), SEQ ID NO:4 (FAO2a protein),and SEQ ID NO:6 (FAO2b protein), the first signature peptide (SEQ IDNO:13) is located at amino acid positions 355 to 362. A second signaturepeptide has the amino acid sequence IIGSGAGAGVMA (SEQ ID NO:14). Thissecond signature peptide is present in the FOA2a and FAO2b proteins ofthe present invention but not in the FAO1 protein of the presentinvention, nor previously characterized yeast FAO proteins. Withreference to SEQ ID NOs:4 and 6, the second signature peptide is locatedat amino acid positions 198 to 209. A third signature peptide is foundin the FAO2a and FAO2b proteins of the present invention but not inother previously characterized yeast FAO proteins. The third signaturepeptide has the amino acid sequence AGSTLGGG (SEQ ID NO:19). Withreference to SEQ ID NOs:4 and 6, this third signature peptide is locatedat amino acid positions 262 to 269.

Thus in accordance with the present invention, there is provided apeptide having the amino acid sequence: CGFCYLGC (SEQ ID NO:13). Also inaccordance with the present invention, there is provided a peptidehaving the amino acid sequence IIGSGAGAGVMA (SEQ ID NO:14). A peptidehaving the amino acid sequence AGSTLGGG (SEQ ID NO:19) is also providedby the present invention. These peptides are useful e.g., in producingantibodies which specifically bind to a yeast FAO comprising the aminoacid sequence of the signature peptides. Such antibodies are useful inimmunoassays such as radioimmunoassays, enzyme-linked immunosorbentassays (ELISA), Westerns blot, immunofluorescent assays,chemiluminescent assays and bioluminescent assays. These types of assaysare useful for monitoring the FAO enzyme levels at different timesduring a fermentation run and can aid in solving problems that mightarise during fermentation.

Structurally related amino acid sequences may be substituted for thedisclosed sequences set forth in SEQ ID NOs: 2, 4, 6, 10, 12, 13, 14, or19 in practicing the present invention. Amino acid insertionalderivatives of the proteins and peptides of the present inventioninclude amino and/or carboxyl terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Insertional amino acidsequence variants are those in which one or more amino acid residues areintroduced into a predetermined site in a subject FAO protein or peptidealthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants may be made by removing oneor more amino acids from the sequence of a subject peptide.Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different residueinserted in its place. Typical substitutions are those made inaccordance with the following Table 1:

TABLE 1 Suitable residues for amino acid substitutions Original ResidueExemplary Substitutions Ala (A) Ser Arg (R) Lys Asn (N) Gln; His Asp (D)Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Pro His (H) Asn; Gln Ile(I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Ile Phe(F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; PheVal (V) Ile; Leu

When a subject FAO protein or peptide is derivatised by amino acidsubstitution, the amino acids are generally replaced by other aminoacids having like properties such as hydrophobicity, hydrophilicity,electronegativety, bulky side chains and the like. As used herein, theterms “derivative”, “analogue”, “fragment”, “portion” and “likemolecule” refer to a subject FAO protein having an amino acid sequenceas set forth in SEQ ID NOs: 2, 4, 6, 10 or 12 and having an amino acidsubstitution, insertion, addition, or deletion, as long as saidderivative, analogue, fragment, portion, or like molecule retains theability to function as a fatty alcohol oxidase, i.e., retains theability to convert an alcohol to an aldehyde. Likewise, the terms“derivative”, “analogue”, “fragment”, “portion” and “like molecule” mayalso refer to a subject FAO signature peptide having an amino acidsequence as set forth in SEQ ID NOs: 13, 14, or 19 and having an aminoacid substitution, insertion, addition, or deletion, as long as saidderivative, analogue, fragment, portion, or like molecule of an FAOsignature peptide, when incorporated within a larger FAO protein,derivative, analogue, fragment, portion or like-molecule, does notdiminish FAO activity.

The synthetic peptides of the present invention may be synthesized by anumber of known techniques. For example, the peptides may be preparedusing the solid-phase technique initially described by Merrifield (1963)in J. Am. Chem. Soc. 85:2149–2154. Other peptide synthesis techniquesmay be found in M. Bodanszky et al. Peptide Synthesis, John Wiley andSons, 2d Ed., (1976) and other references readily available to thoseskilled in the art. A summary of polypeptide synthesis techniques may befound in J. Sturart and J. S. Young, Solid Phase Peptide Synthesis,Pierce Chemical Company, Rockford, Ill., (1984). Peptides may also besynthesized by solution methods as described in The Proteins, Vol. II,3d Ed., Neurath, H. et al., Eds., pp. 105–237, Academic Press, New York,N.Y. (1976). Appropriate protective groups for use in different peptidesyntheses are described in the texts listed above as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York,N.Y. (1973). The peptides of the present invention may also be preparedby chemical or enzymatic cleavage from larger portions of a subject FAOprotein or from a full length FAO protein.

Additionally, the FAO proteins and peptides of the present invention mayalso be prepared by recombinant DNA techniques. For most amino acidsused to build proteins, more than one coding nucleotide triplet (codon)can code for a particular amino acid residue. This property of thegenetic code is known as redundancy. Therefore, a number of differentnucleotide sequences may code for a particular FAO protein or peptide.

Thus, other FAO proteins or enzymatically active fragments thereof,which share sufficient amino acid identities with the FAO1, FAO2a, andFAO2b of the present invention (SEQ ID NOs: 2, 4, and 6) are within thescope of the present invention. For example, the present inventionprovides an FAO enzyme having an amino acid sequence identity greaterthan 82% when compared to the amino acid sequence of the FAO1 as setforth in SEQ ID NO:2. Preferably, an FAO enzyme having an amino acidsequence identity greater than 82% when compared to the amino acidsequence of the FAO1 set forth in SEQ ID NO:2 comprises the signaturemotif CGFCYLGC (SEQ ID NO:13). In another embodiment, the FAO enzyme hasan amino acid sequence identity greater than 83% when compared to theamino acid sequence of FAO1 as set forth in SEQ ID NO:2. Preferably, anFAO enzyme having an amino acid sequence identity greater than 83% whencompared to the amino acid sequence set forth in SEQ ID NO:2 comprisesthe signature motif CGFCYLGC (SEQ ID NO:13). In still anotherembodiment, an FAO enzyme has an amino acid sequence identity greaterthan 84% when compared to the amino acid sequence of FAO1 as set forthin SEQ ID NO:2. Preferably, an FAO enzyme having an amino acid sequenceidentity greater than 84% when compared to the amino acid sequence setforth in SEQ ID NO:2 comprises the signature motif GGFCYLGC (SEQ IDNO:13). Even more preferably, the FAO enzyme has an amino acid sequenceidentity greater than 85% when compared to the amino acid sequence ofFAO1 as set forth in SEQ ID NO:2. Preferably, an FAO enzyme having anamino acid sequence identity greater than 85% when compared to the aminoacid sequence of FAO1 as set forth in SEQ ID NO:2 comprises thesignature motif GGFCYLGC (SEQ ID NO:13). In a still more preferredembodiment, a subject FAO enzyme has an amino acid sequence identitygreater than 90% when compared to the amino acid sequence of FAO1 as setforth in SEQ ID NO:2. In this embodiment, it is preferable that asubject FAO enzyme having an amino acid identity greater than 90% whencompared to the amino acid sequence of FAO1 as set forth in SEQ ID NO:2also comprises the signature motif GGFCYLGC (SEQ ID NO:13). In a mostpreferred embodiment, the FAO enzyme has an amino acid identity greaterthan 95% when compared to the amino acid sequence of FAO1 as set forthin SEQ ID NO:2. In this embodiment, it is preferable that a subject FAOenzyme having an amino acid identity of greater than 95% when comparedto the amino acid sequence of FAO1 as set forth in SEQ ID NO:2 alsocomprises the signature motif GGFCYLGC (SEQ ID NO:13).

In another embodiment, the present invention provides an FAO enzymehaving an amino acid sequence identity greater than 85% when compared tothe amino acid sequence of FAO2a as set forth in SEQ ID NO:4. In thisembodiment, the subject FAO enzyme preferably also comprises thesignature motif IIGSGAGAGVMA (SEQ ID NO:14) and/or AGSTLGGG (SEQ IDNO:19), and/or GGFCYLGC (SEQ ID NO:13). In another embodiment, a subjectFAO enzyme has an amino acid sequence identity greater than 86% whencompared to the amino acid sequence of the FAO2a as set forth in SEQ IDNO:4. In this embodiment, it is preferred that the subject FAO enzymecomprises the signature motif IIGSGAGAGVMA (SEQ ID NO:14) and/orAGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13). In an even morepreferred embodiment, a subject FAO enzyme has an amino acid sequenceidentity greater than 87% when compared to the amino acid sequence ofthe FAO2a as set forth in SEQ ID NO:4. In this embodiment, a subject FAOenzyme having an amino acid sequence identity greater than 87%preferably also comprises the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).Still more preferably, the FAO enzyme has an amino acid sequenceidentity greater than 88% when compared to the amino acid sequence ofthe FAO2a as set forth in SEQ ID NO:4. In this embodiment, the FAOenzyme having an amino acid sequence identity greater than 88% whencompared to the amino acid sequence of the FAO2a as set forth in SEQ IDNO:4 preferably comprises the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).In an even more preferred embodiment, the FAO enzyme has an amino acidsequence identity greater than 90% when compared to the amino acidsequence of the FAO2a as set forth in SEQ ID NO:4. In this embodiment,it is preferred that the FAO enzyme having an amino acid sequenceidentity greater than 90% when compared to the amino acid sequence ofthe FAO2a as set forth in SEQ ID NO:4 also comprises the signature motifIIGSGAGAGVM (SEQ ID NO:14) and/or AGSTLGGG (SEQ ID NO:19), and/orGGFCYLGC (SEQ ID NO:13). In a most preferred embodiment, the FAO enzymehas an amino acid sequence identity greater than 95% when compared tothe amino acid sequence of the FAO2a as set forth in SEQ ID NO:4. Inthis embodiment, the FAO enzyme having an amino acid sequence identitygreater than 95% when compared to the amino acid sequence of the FAO2aas set forth in SEQ ID NO:4 preferably also comprises the signaturemotif IIGSGAGAGVM (SEQ ID NO:14) and/or AGSTLGGG (SEQ ID NO:19), and/orGGFCYLGC (SEQ ID NO:13).

In still another embodiment, the present invention provides an FAOenzyme having an amino acid sequence identity greater than 85% whencompared to the amino acid sequence of FAO2b as set forth in SEQ IDNO:6. Preferably, an FAO enzyme having an amino acid sequence identitygreater than 85% when compared to the amino acid sequence of FAO2b asset forth in SEQ ID NO:6 also comprises the signature motif IIGSGAGAGVMA(SEQ ID NO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ IDNO:13). In another embodiment, a subject FAO enzyme has an amino acidsequence identity greater than 86% when compared to the amino acidsequence of the FAO2b as set forth in SEQ ID NO:6. In this embodiment,the FAO enzyme having an amino acid sequence identity greater than 86%when compared to the amino acid sequence of the FAO2b as set forth inSEQ ID NO:6 preferably comprises the signature motif IIGSGAGAGVMA (SEQID NO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ IDNO:13). Even more preferably, the FAO enzyme has an amino acid sequenceidentity greater than 87% when compared to the amino acid sequence ofthe FAO2b as set forth in SEQ ID NO:6. In this embodiment, the FAOenzyme having an amino acid sequence identity greater than 87% whencompared to the amino acid sequence of the FAO2b as set forth in SEQ IDNO:6 preferably comprises the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).Still more preferably, the FAO enzyme has an amino acid sequenceidentity greater than 88% when compared to the amino acid sequence ofthe FAO2b as set forth in SEQ ID NO:6. In this embodiment, the FAOenzyme having an amino acid sequence identity greater than 88% whencompared to the amino acid sequence of the FAO2b as set forth in SEQ IDNO:6 preferably also comprises the signature motif IIGSGAGAGVM (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).In an even more preferred embodiment, the FAO enzyme has an amino acidsequence identity greater than 90% when compared to the amino acidsequence of the FAO2b as set forth in SEQ ID NO:6. Preferably, the FAOenzyme having an amino acid sequence identity greater than 90% whencompared to the amino acid sequence of the FAO2b enzyme as set forth inSEQ ID NO:6 also comprises the signature motif IIGSGAGAGVM (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).In a most preferred embodiment, the FAO enzyme has an amino acidsequence identity greater than 95% when compared to the amino acidsequence of the FAO2b as set forth in SEQ ID NO:6. In this embodiment,the FAO enzyme having an amino acid identity greater than 95% whencompared to the amino acid sequence of the FAO2b as set forth in SEQ IDNO:6 also preferably comprises the signature motif IIGSGAGAGVM (SEQ IDNO:14) and/or AGSTLGGG (SEQ ID NO:19), and/or GGFCYLGC (SEQ ID NO:13).

The present invention also provides nucleic acid molecules comprisingnucleotide sequences which code for the signature motif of a subjectFAO1 and FAO2. For example, a nucleotide sequence which encodes thesignature motif CGFCYLGC (SEQ ID NO:13) is provided as:

TGY GGN TTY TGY TAY YTN GGN TGY (SEQ ID NO:32)wherein:

R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is Aor T, H is A or T or C, B is G or T or C, D is G or A or T, N is A or G,or C or T.

In addition, a nucleic acid molecule comprising a nucleotide sequencewhich codes for the signature motif IIGSGAGAGVMA (SEQ ID NO:14) of asubject FAO2 is provided which has the following sequence:

ATH ATH GGN WSN GGN GCN GGN GCN GGN GTN ATG GCN (SEQ ID NO:33)

wherein:

R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is Aor T, H is A or T or C, B is G or T or C, D is G or A or T, N is A or G,or C or T.

Further, a nucleic acid molecule comprising a nucleotide sequence whichcodes for the signature motif AGSTLGGG (SEQ ID NO:19) is provided as:

GCN GGN WSN ACN YTN GGN GGN GGN (SEQ ID NO:34)

wherein R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C,W is A or T, H is A or T or C, B is G or T or C, D is G or A or T, N isA or G, or C or T.

The nucleic acid molecules encoding the FAO1 and FAO2 signature motifsare useful for identifying and isolating genes encoding FAO proteinsfrom Candida tropicalis.

In another aspect of the invention, there are provided nucleotidesequences which have a sequence identity of greater than 77% whencompared to the nucleotide sequence of the subject FAO1 ORF (SEQ IDNO:1) and which also encode the signature motif CGFCYLGC (SEQ ID NO:13).Preferably, the nucleotide sequence has a sequence identity of greaterthan 78% when compared to the nucleotide sequence of the subject FAO1ORF (SEQ ID NO:1) and encodes the signature motif CGFCYLGC (SEQ IDNO:13). More preferably, the nucleotide sequence has a nucleic acidsequence identity of greater than 79% when compared to the nucleotidesequence of the subject FAO1 ORF (SEQ ID NO:1) and encodes the signaturemotif CGFCYLGC (SEQ ID NO:13). Even more preferably, the nucleotidesequence has a sequence identity of greater than 79% when compared tothe nucleotide sequence of the subject FAO1 ORF (SEQ ID NO:1) andencodes the signature motif CGFCYLGC (SEQ ID NO:13). In a most preferredembodiment, the nucleotide sequence has a sequence identity of greaterthan 80% when compared to the nucleotide sequence of the subject FAO1ORF (SEQ ID NO:1) and encodes the signature motif CGFCYLGC (SEQ IDNO:13).

In yet another aspect of the present invention there are providednucleotide sequences which have a sequence identity of greater than 78%when compared to the nucleotide sequence of the subject FAO2a ORF (SEQID NO:3) and which also encode the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).Preferably, the nucleotide sequence has a sequence identity of greaterthan 79% when compared to the nucleotide sequence of the subject FAO2aORF (SEQ ID NO:3) and encodes the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).More preferably, the nucleotide sequence has a sequence identity ofgreater than 80% when compared to the nucleotide sequence of the subjectFAO2 ORF (SEQ ID NO:3) and encodes the signature motif IIGSGAGAGVMA (SEQID NO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).

In yet another aspect of the present invention there are providednucleotide sequences which have a sequence identity of greater than 78%when compared to the nucleotide sequence of the subject FAO2b ORF (SEQID NO:5) and which also encode the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).Preferably, the nucleotide sequence has a sequence identity of greaterthan 79% when compared to the nucleotide sequence of the subject FAO2bORF (SEQ ID NO:5) and encodes the signature motif IIGSGAGAGVMA (SEQ IDNO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).More preferably, the nucleotide sequence has a sequence identity ofgreater than 80% when compared to the nucleotide sequence of the subjectFAO2b ORF (SEQ ID NO:5) and encodes the signature motif IIGSGAGAGVMA(SEQ ID NO:14) and/or CGFCYGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ IDNO:19).

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. Thus, the determination of percentidentity between any two sequences can be accomplished using amathematical algorithm. Computer implementations of these mathematicalalgorithms can be utilized for comparison of sequences to determinesequence identity. Such implementations include, but are not limited to:CLUSTAL in the PC/Gene program (available from Intelligenetics, MountainView, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8(available from Genetics Computer Group (CGC), 575 Science Drive,Madison, Wis., USA). Alignments using these programs can be performedusing the default programs. The CLUSTAL program is well described byHigns et al., 1988, Higgins et al., 1989, Corpet et al. 1988, Huang etal. 1992, and Pearson et al., 1994. Softeware for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information ( ). For purposes of the present invention,comparison of amino acid and nucleotide sequences for determination ofpercent sequence identity to the nucleotide and amino acid sequences ofFAO1 and FAO2, is preferably made using the BlastN program (version1.4.7 or later) with its default parameters or any equivalent program.By “equivalent program” is intended any sequence comparison programthat, for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by the preferred program.

Another way of describing isolated nucleic acid molecules encoding asubject FAO is in terms of hybridization to the coding sequences (ORFs)of the subject FAO1 (SEQ ID NO:2) and FAO2a and FAO2b genes (SEQ ID NOs:4 and 6, respectively). Thus in accordance with the present invention,there is provided a nucleic acid molecule which hybridizes under mediumto high stringency conditions to the coding sequence of the subject FAO1gene i.e., nucleotides 1941–4054 of the nucleotide sequence set forth inSEQ ID NO:1 and which also encodes the signature motif CGFCYLGC (SEQ IDNO:13). Preferably, the nucleic acid molecule which hybridizes undermedium to high stringency conditions to the coding sequence of thesubject FAO1 gene and encodes the signature motif CGFCYLGC (SEQ IDNO:13), comprises the nucleotide sequence:

TGY GGN TTY TGY TAY YTN GGN TGY (SEQ ID NO:32)wherein:

R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is Aor T, H is A or T or C, B is G or T or C, D is G or A or T, N is A or G,or C or T.

The present invention further provides a nucleic acid molecule whichhybridizes under medium to high stringency conditions to the codingsequence of the subject FOA2a gene, i.e., nucleotides 1521–3635 of SEQID NO:3, or FAO2b gene, i.e., nucleotides 1099–3213 of SEQ ID NO:5, andwhich also encodes the signature motif IIGSGAGAGVMA (SEQ ID NO:14)and/or CGFCYLGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19).Preferably, the nucleic acid molecule which hybridizes under medium tohigh stringency conditions to the coding sequence of a subject FAO2a orFAO2b gene and encodes the signature motif IIGSGAGAGVMA (SEQ ID NO:14)and/or and/or CGFCYLGC (SEQ ID NO:13) and/or AGSTLGGG (SEQ ID NO:19),comprises the nucleotide sequence:

(SEQ ID NO:33) ATH ATH GGN WSN GGN GCN GGN GCN GGN GTN AUG GCN and/or(SEQ ID NO:32)         TGY GGN TTY TGY TAY YTN GGN TGY and/or (SEQ IDNO:34)         GCN GGN WSN ACN YTN GGN GGN GGNwherein:

R is A or G, Y is C or T, M is A or C, K is G or T, S is G or C, W is Aor T,H is A or T or C, B is G or T or C, D is G or A or T, N is A or G,or C or T.

As used herein, hybridization under medium or high stringency conditionsare as described in Maniatis et al. 1982 Molecular Cloning, Cold SpringHarbor Laboratory, N.Y., at pages 387–389, and especially pragraph 11which is incorporated by reference herein as if fully set forth. A lowstringency is defined as being in 4–6 S×SSC/1% (w/v) SDS at 37–45° C.for 2–3 hours. Medium stringency conditions are considered herein to be1–4×SSC/0.5%–1% (w/v) SDS at greater than or equal to 45° C. for 2–3hours. High stringency conditions are considered herein to be0.1–1×SSC/0.1%–1% (w/v) SDS at greater than or equal to 60° C. for 1–3hours. As used herein, medium to high stringency conditions refer toconditions which are either medium stringency conditions, highstringency conditions, or conditions between medium and high stringency.Preferably, an isolated nucleic acid molecule which hybridizes to theORF of the subject FAO1 or FAO2 genes, hybridizes under high stringencyconditions.

The present invention further provides a vector comprising a nucleotidesequence encoding FAO1 having the amino acid sequence as set forth inSEQ ID NO:2. In a preferred embodiment, the vector comprises an FAO1gene having a nucleotide sequence as set forth in SEQ ID NO:1. Alsoprovided is a vector comprising a nucleotide sequence encoding FAO2ahaving the amino acid sequence as set forth in SEQ ID NO:4. Preferably,the vector comprises an FAO2a gene having a nucleotide sequence as setforth in SEQ ID NO:3. Still further provided is a vector comprising anucleotide sequence encoding FAO2b having the amino acid sequence as setforth in SEQ ID NO:6. Preferably, the vector comprises an FAO2b genehaving a nucleotide sequence as set forth in SEQ ID NO:5.

In accordance with the present invention, any of the nucleic acidsequences hereinbefore described may be incorporated into a vector. Inaddition, it should be understood that a vector may also comprise anopen reading frame for both an FAO1 and FAO2 gene, as well as fragmentsthereof.

A host cell is also provided which is transfected or transformed with anucleic acid molecule encoding a subject FAO1 and/or FAO2a and/or FAO2b.For example, a host cell may be transfected or transformed with anucleic acid molecule encoding an amino acid sequence as set forth inany of SEQ ID NOs: 2, 4, or 6. Preferably, the host cell is transformedwith a nucleic acid molecule encoding an FAO1 gene comprising thenucleotide sequence depicted in SEQ ID NO:1. In another preferredembodiment, the host cell is transformed with a nucleic acid moleculeencoding an FAO2a gene comprising the nucleotide sequence depicted inSEQ ID NO:3. In still another preferred embodiment, the host cell istransformed with a nucleic acid molecule encoding an FAO2b genecomprising the nucleotide sequence depicted in SEQ ID NO:5. It should beunderstood that the present invention also encompasses host cellstransfected or transformed with any of the nucleic acid sequenceshereinbefore described.

Depending upon the host cell transformed with a subject FAO2a or FAO2bgene, the CUG codon in mRNA corresponding to nucleotide positions2049–2051 of SEQ ID NOs: 3 and 5 will be translated differently. Forexample, while CUG codons are translated as serine in C. tropicalisother host organisms follow the universal code and translate CUG codonsas leucine. Therefore, in order to ensure that the mRNA codoncorresponding to nucleotide positions 2049–2051 of SEQ ID No:3 andnucleotide positions 1627–1629 in SEQ ID No:5 in a subject FAO2 gene istranslated to serine in a host cell other than C. tropicalis, such codonshould be one which is translated to serine following the universalcode. Examples of such codons include UCU, UCC, UCA, UCG, AGU, and AGC.Methods of altering DNA sequence (and therefore codons in correspondingtranscribed mRNA sequence) are well known in the art and include e.g.,various in vitro mutagenesis techniques. There are various commerciallyavailable kits particularly suited for this application such as theT7-Gen in vitro mutagenesis Kit (USB, Cleveland, Ohio). Alternatively,PCR technology may be employed to alter nucleotide sequence. If an FAO2gene is chemically synthesized, nucleotide sequence corresponding topositions 2049–2051 of SEQ ID NOs: 3 and 5 can be synthesized as DNAwhich transcribes a codon translatable to serine following the universalcode, e.g., incorporating DNA sequence corresponding to those codonslisted above.

In a preferred embodiment of the invention, an isolated nucleic acidmolecule which encodes FAO1 is manipulated so that the native FAO1 genepromoter is removed and a promoter from another gene is operably linkedto the FAO1 gene coding sequence. Similarly, an isolated nucleic acidmolecule which encodes FAO2 is preferably manipulated so that the nativeFAO2 gene promoter is removed and a promoter from another gene isoperably linked to the FAO2 gene coding sequence. The term “operablylinked” refers to the association of nucleic acid sequences so that thefunction of one is affected by the other. A promoter is operably linkedwith an open reading frame of a gene, when it is capable of affectingthe expression of the open reading frame (ORF) (i.e., the ORF is underthe transcriptional control of the promoter.) Notwithstanding thepresence of other sequences between the promoter and the ORF, it shouldbe understood that a promoter may still be operably linked to the ORF.

Desirable promoters for substitution into an FAO1 or FAO2 gene includepromoters which may be induced at various times during bioconversion inresponse to certain stimuli (e.g., stress, substrate, cell death)thereby leading to increased aldehyde production and increaseddicarboxylic acid production at defined times during the bioprocess.Examples of promoters suitable for operably linking to the FAO1 or FAO2open reading frames include e.g., CYP52A2A, CYP52A5A, and CYP52A1A (seeU.S. Pat. No. 6,331,420, the disclosure of which is incorporated byreference herein as if fully set forth.) With respect to use of theCYP52A2A promoter, see also copending patent application Ser. No.09/911,781, the disclosure of which is also incorporated by referenceherein as if fully set forth. The CYP52A2A gene of C. tropicalis 20336is one gene from a family of genes involved in the metabolism of oleicacid to produce oleic dicarboxylic acid. The level of transcriptionalinduction of this gene in an oleic acid fermentation is many fold (>25)above other members of the same family. Example 6 herein describes themaking of fusions between the CYP52A2A gene promoter and the ORF of FAO1and FAO2 genes. Promoters from Candida β-oxidation genes such as POX 4or POX 5 may also be operably linked to an ORF frame of an FAO1 or FAO2gene. Preferably, a CYP52A2A gene promoter is used to drive expressionof an FAO1 or FAO2 gene.

Thus, in accordance with the present invention, there is provided anucleotide sequence for an open reading frame (ORF) of a gene encodingan FAO having the amino acid sequence as set forth in SEQ ID NO:2,wherein the ORF is operably linked to a heterologous promoter.Preferably, a nucleotide sequence for an ORF encoding an FAO operablylinked to a heterologous promoter comprises nucleotides 1941–4054 of thenucleotide sequence set forth in SEQ ID NO:1.

Similarly, there is provided a nucleotide sequence for an open readingframe (ORF) of an FAO having the amino acid sequence as set forth in SEQID NO:4 (FAO2a) or SEQ ID NO:6 (FAO2b), wherein the ORF is operablylinked to a heterologous promoter. Preferably, a nucleotide sequence foran ORF encoding an FAO operably linked to a heterologous promotercomprises either nucleotides 1521–3635 of the nucleotide sequence setforth in SEQ ID NO:3 or nucleotides 1099–3213 of the nucleotide sequenceset forth SEQ ID NO:5. As used herein, the term “heterologous promoter”is meant to include a promoter other than the native promoter associatedwith a particular FAO coding sequence. These nucleotide sequences cantherefore also be described as chimeric genes.

In another aspect of the invention, there is provided an expressionvector comprising a nucleotide sequence encoding an FAO1 or FAO2 geneoperably linked to a heterologous promoter sequence. The term“expression vector” is used broadly herein and is intended to encompassany medium which includes a nucleic acid molecule and which can be usedto transform a target cell. Expression vectors thus encompass all theexamples of vectors listed herein including integration vectors.Examples of expression vectors include but are not limited to plasmids,phagemids, phage, cosmids, yeast artificial chromosomes or linear DNAvectors. Examples of plasmids include but are not limited to e.g., yeastepisomal plasmids or yeast replication plasmids.

A method of producing an FAO1 protein including an amino acid sequenceas set forth in SEQ ID NO:2 is also provided. The method comprises thesteps of (a) transforming a suitable host cell with a DNA sequence thatencodes a protein having the amino acid sequence as set forth in SEQ IDNO:2; and (b) culturing the cell under conditions favoring theexpression of the protein.

A method of producing an FAO2a protein including an amino acid sequenceas set forth in SEQ ID NO:4 is also provided. The method comprises thesteps of (a) transforming a suitable host cell with a DNA sequence thatencodes a protein having the amino acid sequence as set forth in SEQ IDNO:4; and (b) culturing the cell under conditions favoring expression ofthe protein.

A method of producing an FAO2b protein including an amino acid sequenceas set forth in SEQ ID NO:6 is also provided. The method comprises thesteps of (a) transforming a suitable host cell with a DNA sequence thatencodes a protein having the amino acid sequence as set forth in SEQ IDNO:6; and (b) culturing the cell under conditions favoring theexpression of the protein

In another aspect of the invention, there is provided a method forincreasing production of an aldehyde during the second step of theω-oxidation pathway of fatty acids. The method comprises the steps of:(a) providing a host cell having a naturally occurring number of FAOgenes; (b) increasing, in the host cell, the number of FAO1 genes whichencode an FAO1 protein having the amino acid sequence as set forth inSEQ ID NO:2; and (c) culturing the host cell in media containing anorganic substrate which upregulates the FAO1 gene, to effect increasedproduction of an aldehyde. Preferably, the FAO1 gene comprisesnucleotides 1941–4054 of the nucleotide sequence as set forth in SEQ IDNO:1.

A method for increasing production of an aldehyde during the second stepof the ω-oxidation pathway of fatty acids may also comprise the stepsof: (a) providing a host cell having a naturally occurring number of FAOgenes; (b) increasing, in the host cell, the number of FAO2 genes whichencode an FAO2a protein having the amino acid sequence as set forth inSEQ ID NO:4; and (c) culturing the host cell in media containing anorganic substrate which upregulates the FAO2 gene, to effect increasedproduction of an aldehyde. Preferably, the FAO2a gene comprisesnucleotides 1521–3635 of the nucleotide sequence as set forth in SEQ IDNO:3.

Alternatively, a method for increasing production of an aldehyde duringthe second step of the ω-oxidation pathway of fatty acids comprises thesteps of: (a) providing a host cell having a naturally occurring numberof FAO genes; (b) increasing, in the host cell, the number of FAO2 geneswhich encode an FAO2b protein having the amino acid sequence as setforth in SEQ ID NO:6; and (c) culturing the host cell in mediacontaining an organic substrate which upregulates the FAO2b gene, toeffect increased production of an aldehyde. Preferably, the FAO2b genecomprises nucleotides 1099–3213 of the nucleotide sequence as set forthin SEQ ID NO:5.

In another aspect of the invention, there is provided a method forincreasing production of a dicarboxylic acid. The method comprises thesteps of: (a) providing a host cell having a naturally occurring numberof FAO genes; (b) increasing, in the host cell, the number of FAO1 geneswhich encode an FAO1 protein having the amino acid sequence as set forthin SEQ ID NO:2; and (c) culturing the host cell in media containing anorganic substrate which upregulates the FAO1 gene, to effect increasedproduction of dicarboxylic acid. Preferably, the FAO1 gene comprisesnucleotides 1941–4054 of the nucleotide sequence as set forth in SEQ IDNO:1.

Alternatively, a method for increasing production of a dicarboxylic acidcomprises the steps of: (a) providing a host cell having a naturallyoccurring number of FAO genes; (b) increasing, in the host cell, thenumber of FAO2a genes which encode an FAO2 protein having the amino acidsequence as set forth in SEQ ID NO:4; and (c) culturing the host cell inmedia containing an organic substrate which upregulates the FAO2a gene,to effect increased production of dicarboxylic acid. Preferably, theFAO2a gene comprises nucleotides 1521–3635 of the nucleotide sequence asset forth in SEQ ID NO:3.

Alternatively, a method for increasing production of a dicarboxylic acidcomprises the steps of: (a) providing a host cell having a naturallyoccurring number of FAO genes; (b) increasing, in the host cell, thenumber of FAO2b genes which encode an FAO2 protein having the amino acidsequence as set forth in SEQ ID NO:6; and (c) culturing the host cell inmedia containing an organic substrate which upregulates the FAO2b gene,to effect increased production of dicarboxylic acid. Preferably, theFAO2b gene comprises nucleotides 1099–3213 of the nucleotide sequence asset forth in SEQ ID NO:5.

A method for increasing the production of an FAO1 protein having anamino acid sequence as set forth in SEQ ID NO:2 is further provided. Themethod comprises the steps of: (a) transforming a host cell having anaturally occurring level of FAO1 protein with an increased copy numberof an FAO1 gene that encodes the FAO1 protein having the amino acidsequence as set forth in SEQ ID NO:2 and (b) culturing the cell andthereby increasing expression of the protein compared with that of ahost cell containing a naturally occurring copy number of the FAO1 gene.

A method for increasing the production of an FAO2 protein having anamino acid sequence as set forth in SEQ ID NO:4 or 6 is also provided.The method comprises the steps of: (a) transforming a host cell having anaturally occurring amount of FAO2 protein with an increased copy numberof an FAO2 gene that encodes the FAO2 protein having the amino acidsequence as set forth in SEQ ID NO:4 or 6; and (b) culturing the celland thereby increasing expression of the protein compared with that of ahost cell containing a naturally occurring copy number of the FAO2 gene.

In addition to amplifying the FAO genes to improve diacid productivityand decrease ω-hydroxy fatty acid formation, the FAO genes can be usedto create knockout constructs to disrupt the native FAO genes in Candidatropicalis. Methods for making knockout constructs and use of the samefor gene disruption are well known in the art. The disruption of nativeFAO genes in C. tropicalis block the diacid pathway at that step andallow for a build-up of α,ω-dihydroxy compounds when utilizing alkanesubstrates and ω-hydroxy fatty acids when using fatty acid substrates.

The present invention provides additional methods for increasingproduction of an aldehyde from an alcohol during the second step of theω-oxidation pathway of fatty acids and ultimately, for increasingproduction of dicarboxylic acid. For example, a method for increasingproduction of an aldehyde during the second step of the ω-oxidationpathway of fatty acids comprises the steps of: (a) isolating a nucleicacid molecule comprising coding sequence for an FAO1 gene; (b) isolatinga promoter sequence from a gene which transcribes at a higher rate thanthe FAO1 gene; (c) operably linking the promoter sequence to the openreading frame (ORF) of the FAO1 gene to create a fusion gene; (d)inserting the fusion gene into an expression vector; (e) transforming ahost cell with the expression vector and (f) culturing the transformedhost cell in a media containing an organic substrate that isbiooxidizable to a mono- or polycarboxylic acid. Preferably, the nucleicacid molecule encodes an FAO1 comprising the amino acid sequence setforth in SEQ ID NO:2. Preferably, the organic substrate is an ω-hydroxyfatty acid.

Production of an aldehyde from an alcohol during the second step of theω-oxidation pathway of fatty acids may also be increased by: (a)isolating a nucleic acid molecule comprising coding sequence for an FAO2gene; (b) isolating a promoter sequence from a gene which transcribes ata higher rate than the FAO2 gene, (c) operably linking the promotersequence to the open reading frame (ORF) of the FAO2 gene to create afusion gene; (d) inserting the fusion gene into an expression vector;(e) transforming a host cell with the expression vector and (f)culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the FAO2 nucleic acid molecule encodes an FAO2 proteincomprising the amino acid sequence as set forth in SEQ ID NOs:4 or 6.

Production of an ketone from an alcohol during the second step of theω-oxidation pathway of fatty acids may also be increased by: (a)isolating a nucleic acid molecule comprising coding sequence for an FAO2gene; (b) isolating a promoter sequence from a gene which transcribes ata higher rate than the FAO2 gene, (c) operably linking the promotersequence to the open reading frame (ORF) of the FAO2 gene to create afusion gene; (d) inserting the fusion gene into an expression vector;(e) transforming a host cell with the expression vector and (f)culturing the transformed host cell in a media containing an organicsubstrate that is biooxidizable to a mono- or polycarboxylic acid.Preferably, the organic substrate is a 2-alkanol. Preferably, thenucleic acid molecule encodes an FAO2 protein comprising the amino acidsequence as set forth in SEQ ID NOs:4 or 6.

In accordance with the methods of the present invention, dicarboxylicacid production may be increased by: (a) isolating a nucleic acidmolecule comprising coding sequence for an FAO1 gene; (b) isolating apromoter sequence from a gene which transcribes at a higher rate thanthe FAO1 gene; (c) operably linking the promoter sequence to the openreading frame (ORF) of the FAO1 gene to create a fusion gene; (d)inserting the fusion gene into an expression vector; (e) transforming ahost cell with the expression vector and (f) culturing the transformedhost cell in a media containing an organic substrate that isbiooxidizable to a mono- or polycarboxylic acid. Preferably, the ORF ofthe FAO1 gene comprises nucleotides 1941–4054 of the nucleotide sequenceset forth in SEQ ID NO:1. Preferably, the organic substrate is anω-hydroxy fatty acid.

Dicarboxylic acid production may also be increased by: (a) isolating anucleic acid molecule comprising coding sequence for an FAO2 gene; (b)isolating a promoter sequence from a gene which transcribes at a higherrate than the FAO2 gene, (c) operably linking the promoter sequence tothe open reading frame (ORF) of the FAO2 gene to create a fusion gene;(d) inserting the fusion gene into an expression vector; (e)transforming a host cell with the expression vector and (f) culturingthe transformed host cell in a media containing an organic substratethat is biooxidizable to a mono- or polycarboxylic acid. Preferably, theORF of the FAO2 gene comprises nucleotides 1521–3635 of the nucleotidesequence as set forth in SEQ ID NO:3 or nucleotides 1099–3213 of thenucleotide sequence set forth in SEQ ID NO:5.

It is to be understood, that in any of the methods disclosed herein,FAO1 and FAO2 may be separately produced in a host cell. Alternatively,both FAO1 and FAO2 may be produced in the same host cell. Thus forexample, a host cell may be transformed with: an expression vectorcomprising an FAO1 gene or fragment thereof, an expression vectorcomprising an FAO2a gene or fragment thereof, an expression vectorcomprising an FAO2b gene or fragment thereof, an expression vectorcomprising both an FAO1 and/or and FAO2a gene or fragment thereof and/or an FAO2b gene or fragment(s) thereof, or any combination of suchvectors.

It should be understood that host cells into which one or more copies ofdesired FAO1 and/or FAO2 genes (including FAO chimeric genes comprisingan FAO1 or FAO2 gene operably linked to a heterologous promoter) havebeen introduced can be made to include such genes by any technique knownto those skilled in the art. For example, suitable host cells includeprocaryotes such as Bacillus sp., Pseudomous sp., Actinomycetes sp.,Eschericia sp., Mycobacterium sp., and eukaryotes such as yeast, algae,insect cells, plant cells and filamentous fungi. Suitable host cells arepreferably yeast cells such as Yarrowia, Bebaromyces, Saccharomyces,Schizosaccharomyces, and Pichia and more preferably those of the Candidagenus. Preferred species of Candida are tropicalis, maltosa, apicola,paratropicalis, albicans, cloacae, guillermondii, intermedia,lipolytica, parapsilosis and zeylenoides. Particularly preferred hostsinclude C. tropicalis strains that have been genetically modified sothat one or more of the chromosomal POX4A, POX4B and both POX5 geneshave been disrupted as described, e.g., in U.S. Pat. Nos. 5,254,466 and5,620,878, each incorporated herein by reference as if fully set forth.Such disruption blocks the β-oxidation pathway. Examples of β-oxidationblocked strains of C. tropicalis include H41, H41B, H51, H45, H43, H53,H534, H534B, H435 and H5343 (ATCC 20962) as described in aforementionedU.S. Pat. No. 5,254,466.

Vectors such as plasmids, phagemids, phages or cosmids can be used totransform or transfect suitable host cells. Host cells may also betransformed by introducing into a cell a linear DNA vector(s) containingthe desired gene sequence. Such linear DNA may be advantageous when itis desirable to avoid introduction of non-native (foreign) DNA into thecell. For example, DNA consisting of a desired target gene(s) flanked byDNA sequences which are native to the cell can be introduced into thecell by electroporation, lithium acetate transformation, spheroplastingand the like. Flanking DNA sequences can include selectable markersand/or other tools for genetic engineering.

A suitable organic substrate for use in the methods described herein canbe any organic compound that is biooxidizable to a mono- orpolycarboxylic acid (or any compound that is biooxidizable to a ketonegroup for the methods described herein directed to production of aketone). Such a compound can be any saturated or unsaturated aliphaticcompound or any carbocyclic or heterocyclic aromatic compound having atleast one terminal methyl group, a terminal carboxyl group and/or aterminal functional group which is oxidizable to a carboxyl group bybiooxidation. A terminal functional group which is a derivative of acarboxyl group may be present in the substrate molecule and may beconverted to a carboxyl group by a reaction other than biooxidation. Forexample, if the terminal group is an ester that neither the wild-type C.tropicalis nor the genetic modifications described herein will allowhydrolysis of the ester functionality to a carboxyl group, then a lipasecan be added during the fermentation step to liberate free fatty acids.Suitable organic substrates include, but are not limited to, saturatedfatty acids, unsaturated fatty acids, alkanes, alkenes, alkynes andcombinations thereof.

Alkanes are a type of saturated organic substrate which are usefulherein. The alkanes can be linear or cyclic, branched or straight chain,substituted or unsubstituted. Particularly preferred alkanes are thosehaving from about 4 to about 25 carbon atoms, examples of which includebut are not limited to butane, hexane, octane, nonane, dodecane,tridecane, tetradecane, octadecane and the like.

Examples of unsaturated organic substrates which can be used hereininclude but are not limited to internal olefins such as 2-pentene,2-hexene, 3-hexene, 9-octadecene and the like; alpha olefins such as1-dodecene, 1-octadecene, 1-tetradecene, and the like; unsaturatedcarboxylic acids such as 2-hexenoic acid and esters thereof, oleic acidand esters thereof including triglyceryl esters having a relatively higholeic acid content, erucic acid and esters thereof including triglycerylesters having a relatively high erucic acid content, ricinoleic acid andesters thereof including triglyceryl esters having a relatively highricinoleic acid content, linoleic acid and esters thereof includingtriglyceryl esters having a relatively high linoleic acid content;unsaturated alcohols such as 3-hexen-1-ol, 9-octadecen-1-ol, saturatedalcohols such as 2-decanol, 2-undecanol, 2-dodecanol, 2-hexadecanol,10-undecen-1-ol; 1,2-octanediol; 1,10--decanediol; 1,2-dodecanediol;1,16-hexadecanediol; 10-hydroxydecanoic acid; 12-hydroxydodecanoic acid;16-hydroxydodecanoic acid and the like; unsaturated aldehydes such as3-hexen-1-al, 9-octadecen-1-al and the like. In addition to the above,an organic substrate which can be used herein include alicycliccompounds having at least one internal carbon-carbon double bond and atleast one terminal ethyl group, and/or a terminal functional group whichis oxidizable to a carboxyl group by biooxidation.

The organic substrate can also contain other functional groups that arebiooxidizable to carboxyl groups such as an aldehyde or alcohol group.The organic substrate can also contain other functional groups that arenot biooxidizable to carboxyl groups and do not interfere with thebiooxidation such as halogens, ethers, and the like.

Examples of saturated fatty acids which may be applied to cellsincorporating the present FAO1 and FAO2 genes include caproic, enanthic,caprylic, pelargonic, capric, undecylic, lauric, myristic,pentadecanoic, palmitic, margaric, stearic, arachidic, behenic acids andcombinations thereof. Examples of unsaturated fatty acids which may beapplied to cells incorporating the present FAO1 and FAO2 genes includepalmitoleic, oleic, erucic, linoleic, linolenic acids and combinationsthereof. Alkanes and fractions of alkanes may be applied which includechain links from C12 to C24 in any combination. An example of apreferred fatty acid mixture is High Oleic Sun Flower Fatty Acid(HOSFFA). HOSFFA is a fatty acid mixture containing approximately 80%oleic acid and is commercially available from Cognis Corporation asEdenor®. Emersol® is another HOSFFA commercially available from CognisCorporation.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

EXAMPLE 1 Materials and Methods

Transformation of C. tropicalis Using Lithium Acetate

The following protocol was used to transform C. tropicalis in accordancewith the procedures described in Current Protocols in Molecular Biology,Supplement 5, 13.7.1 (1989) Frederick M. Ausubel, Roger Brent, Robert E.Kingston, David D. Moore, J. G. Seidman, John A. Smith, and KevinStruhl, eds., John Wiley and Sons, Hoboken, N.J. 5 ml of YEPD wasinoculated with C. tropicalis H5343 ura- and incubated overnight on aNew Brunswick shaker at 30° C. and 170 rpm. The next day, the overnightculture was used to inoculate 50 ml YEPD at an OD₆₀₀ of 0.2 and growthwas continued at 30° C., 170 rpm. The cells were harvested at an OD₆₀₀of 1.0. The culture was transferred to a 50 ml polypropylene tube andcentrifuged at 1000×g for 10 min. The cell pellet was resuspended in 10ml sterile TE (10 mM Tris-Cl and 1 mM EDTA, pH 8.0). The cells wereagain centrifuged at 1000×g for 10 min and the cell pellet wasresuspended in 10 ml of a sterile lithium acetate solution (LiAc (0.1 Mlithium acetate, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA). Followingcentrifugation at 1000×g for 10 min., the pellet was resuspended in 0.5ml LiAc. This solution was incubated for 1 hr at 30° C. while shakinggently at 50 rpm. A 0.1 ml aliquot of this suspension was incubated with15–20 μg of transforming DNA at 30° C. with no shaking for 30 min. A 0.7ml PEG solution (40% wt/vol polyethylene glycol 3340, 0.1 M lithiumacetate, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) was added and incubated at30° C. for 45 min. The tubes were then placed at 42° C. for 10 min. A0.2 ml aliquot was plated on synthetic complete media minus uracil(SC−uracil) (Kaiser et al. Methods in Yeast Genetics, Cold Spring HarborLaboratory Press, USA, 1994). Growth of transformants was monitored for5 days. After three days, several transformants were picked andtransferred to SC−uracil plates for genomic DNA preparation andscreening.

Fermentations

Fermentations were performed using High Oleic Sun Flower Fatty Acid(HOSFFA) as the substrate, with glucose used as the co-substrate. TheHOSFFA used was approximately 85% oleic acid (84.45% oleic acid, 5.24%linoleic acid, 4.73% stearic acid, 3.87% palmitic acid, 1.71% otherfatty acids). Fermentation runs had both a growth and a bioconversionphase. Cells were grown on glucose at 35° C. to an absorbance at 600 nmof 50 to 60. When the growth phase was completed as determined by asharp rise in dissolved oxygen, the cells were immediately switched tothe bioconversion phase, which was performed at a temperature of 30° C.A slow glucose feed (0.87 g/L/h from 0 to 42 h and 0.7 g/L/h thereafter)was used to supply energy. At the same time a small charge of HOSFFA(0.55% v/v) was added to the fermentation. Feeding of substrate (averagefeed rate of 1.6 g/L/h) continued during the remainder of thefermentation. H5343 is the base strain derived from Candida tropicalisATCC 20336) blocked for β-oxidation, which has had the POX4 and POX5genes disrupted by insertional mutagenesis. Strain HDC23-3 is an H5343base strain that is amplified for a cytochrome P450 monoxygenase gene,CYP52A2, and the cytochrome P450 reductase (NCP) gene, both genes havingbeen cloned from C. tropicalis ATCC 20336.

FAO Activity Profile during HOSFFA Fermentations

Washing of Cells

HDC23-3 cells from several fermentation runs, and taken at differenttime points in the fermentation runs, were prepared and assayed for FAOactivity. Due to the high levels of solid diacid in the broth,particularly in samples taken later in the fermentation, the samples hadto be washed extensively to remove the diacid prior to making extracts.Since frozen samples were found to lose a lot of enzyme activity (afterwashing and preparation of extracts), freshly sampled fermentation brothwas placed on ice until ready for washing. This sample was thoroughlymixed and a 20 ml sample was removed. This sample was centrifuged(Sorvall RT6000B Refrigerated Centrifuge) at approximately 1,500×g(H1000B rotor at 2500 rpm) for 5 minutes to pellet the cells. Thesupernatant was decanted, and the cells were resuspended in 40 ml of 50mM HEPES buffer, pH 7.6. The sample was centrifuged at 1,500×g for onlyone minute, which pelleted the yeast cells, but allowed the less densediacid product to “float” in the supernatant. The supernatant wasdecanted and the cells were washed again in 40 ml of buffer. After thesecond washing step, the cell pellet was examined for any sign ofresidual diacid, evidenced by a white precipitate on top of the cellpellet, or patches of white color in the cell pellet itself. Washing wasrepeated until the pellet was completely clear of all diacid and thecell pellet was a pale tan color throughout. During the course of thefermentation, as the cells produced more diacid, more washing steps wereneeded to free the cell pellet of diacid.

Preparation of Cell-Free Extracts

After washing away the diacid, the cell pellet was resuspended in 10 mlof 50 mM potassium phosphate buffer with 20% glycerol, pH 7.6(phosphate-glycerol buffer) for a two-fold concentration of cellularenzymes. 100 μl of a 100 mM solution of the serine-protease inhibitor,phenylmethylsulfonyl fluoride (PMSF) in isopropanol, was added for afinal concentration of 1 mM. The cells were then broken by passing thesample three times through a chilled French pressure cell (SLMInstruments, Inc. French Pressure Cell Press) at approximately 20,000psig. The sample was stored on ice before, during and after breakage ofthe cells to prevent the loss of enzyme activity. This broken cellsuspension was then centrifuged at approximately 37,000×g for 30 minutesto pellet the cellular debris, leaving the cellular enzymes in thesupernatant. The supernatant (cell-free extract) was removed and savedto perform enzyme assays. During assays, this cellular extract wasalways kept on ice, and was stored at −20° C. between sets of assays topreserve enzyme activity.

FAO Enzyme Assay

The assay procedure used is a modification of an assay used by Kemp etal (1). The assay is a two-enzyme coupled reaction. Dodecanol was usedas the substrate for the FAO, which oxidizes the dodecanol to dodecanal.At the same time it reduces molecular oxygen to hydrogen peroxide. Asecond enzyme in the reaction mixture, peroxidase derived fromhorseradish (HRP), uses electrons obtained by oxidizing hydrogenperoxide to reduce 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) in the reaction mixture. Reduced ABTS absorbs strongly at 405 nm.The quality of HRP is important, since it has been observed thatcommercially obtained preparations vary in quality. It is necessary,therefore, to obtain preparations of highest purity. The HRP (Sigma#P8415) was made at a concentration of 2 mg/ml (≈250 units/mg) in 50 mMpotassium phosphate buffer, pH 7.6. In experiments in which the amountof horseradish peroxidase was varied in the reaction mixture, it wasfound that 5 μl of this solution in a one ml reaction mixture wassufficient to obtain maximal velocity.

The final reaction mixture (1.0 ml) for the general alcohol oxidaseassay consisted of: 500 μl of 200 mM HEPES buffer, pH 7.6; 50 μl of a 10mg/ml ABTS solution in deionized water; 10 μl of a 5 mM solution ofdodecanol in acetone; and 5 μl of a 2 mg/ml horseradish peroxidasesolution in 50 mM potassium phosphate buffer, pH 7.6. After adding theextract, the activity was generally read at 405 nm for one minute atroom temperature. The amount of extract that was added to the reactionmixture was varied so that the activity fell within the range of 0.2 to1.0 ΔA^(405 nm)/min. Alcohol oxidase activity was reported as specificactivity units/mg protein (1 unit=μmole substrate oxidized/min). Anextinction coefficient at 405 nm of 18.4 was used for ABTS and wasequivalent to 0.5 mM oxidized substrate. In certainsubstrate-specificity experiments, 200 mM HEPES buffer, pH 7.6,containing 0.5% Triton X100 was used in place of the 200 mM HEPES bufferin the reaction mixture described above. The detergent aided insolubilizing some of the more water-insoluble substrates tested.

Preparation of Microsomes

Microsomes were prepared from Candida tropicalis cell-free extracts bycentrifugation at 100,000×g for one hour at 4° C. The FAO was foundwithin the pelleted microsomes. Catalase, a soluble enzyme, remained inthe supernatant, which allowed the separation of these two enzymes. Thesupernatant was removed and assayed for FAO and catalase activity. Themicrosomal pellet was resuspended in an equivalent amount ofphosphate-glycerol buffer, after which it was also assayed for catalaseand FAO activity. Microsomes from Escherichia coli were prepared in thesame manner.

Protein Determination

Protein concentration in the extracts was determined. The Lowry methodof determining protein concentration gave much higher results thanexpected, and were inconsistent between protein determinations of thesame samples. For this reason, a new procedure was performed using theBradford Reagent (Sigma, #B6916) and following the protocol provided bythe supplier.

EXAMPLE 2 Cloning of Fatty Alcohol Oxidase Genes

Preparation of Genomic DNA

50 ml of YPD broth (Difco) was inoculated with a single colony of C.tropicalis 20336 from a YPD agar (Difco) plate and was grown overnightat 30° C. 5 ml of the overnight culture was inoculated into 100 ml offresh YPD broth and incubated at 30° C. for 4 to 5 hr with shaking.Cells were harvested by centrifugation, washed twice with steriledistilled water and resuspended in 4 ml of spheroplasting buffer (1 MSorbitol, 50 mM EDTA, 14 mM mercaptoethanol) and incubated for 30 min at37° C. with gentle shaking. 0.5 ml of 2 mg/ml zymolyase (ICNPharmaceuticals, Inc., Irvine, Calif.) was added and incubated at 37° C.with gentle shaking for 30 to 60 min. Spheroplast formation wasmonitored by SDS lysis. Spheroplasts were harvested by briefcentrifugation (4,000 rpm, 3 min) and were washed once with thespheroplast buffer without mercaptoethanol. Harvested spheroplasts werethen suspended in 4 ml of lysis buffer (0.2 M Tris/pH 8.0, 50 mM EDTA,1% SDS) containing 100 mg/ml RNase (Qiagen Inc., Chatsworth, Calif.) andincubated at 37° C. for 30 to 60 min.

Proteins were denatured and extracted twice with an equal volume ofchloroform/isoamyl alcohol (24:1) by gently mixing the two phases byhand inversions. The two phases were separated by centrifugation at10,000 rpm for 10 min and the aqueous phase containing thehigh-molecular weight DNA was recovered. NaCl was added to the aqueouslayer to a final concentration of 0.2 M and the DNA was precipitated byadding 2 volumes of ethanol. Precipitated DNA was spooled with a cleanglass rod and resuspended in TE buffer (10 mM Tris/pH 8.0, 1 mM EDTA)and allowed to dissolve overnight at 4° C. To the dissolved DNA, RNasefree of any DNase activity (Qiagen Inc., Chatsworth, Calif.) was addedto a final concentration of 50 mg/ml and incubated at 37° C. for 30 min.Then protease (Qiagen Inc., Chatsworth, Calif.) was added to a finalconcentration of 100 mg/ml and incubated at 55 to 60° C. for 30 min. Thesolution was extracted once with an equal volume ofphenol/chloroform/isoamyl alcohol (25:24:1) and once with equal volumeof chloroform/isoamyl alcohol (24:1). To the aqueous phase 0.1 volumesof 3 M sodium acetate and 2 volumes of ice cold ethanol (absolute) wereadded and the high molecular weight DNA was spooled with a glass rod anddissolved in 1 to 2 ml of TE buffer.

Library Preparation

A genomic library was constructed using λ ZAP Express™ vector(Stratagene, La Jolla, Calif.). Genomic DNA was partially digested withSau3A1 and fragments in the range of 6 to 12 kb were purified from anagarose gel after electrophoresis of the digested DNA. These DNAfragments were then ligated to BamHI digested λ ZAP Express™ vector armsaccording to manufacturer's protocols. Three ligations were set up toobtain approximately 9.8×10⁵ independent clones. All three librarieswere pooled and amplified according to manufacturer instructions toobtain high-titer (>10⁹ plaque forming units/ml) stock for long-termstorage. The titer of packaged phage library was ascertained afterinfection of E. coli XL1Blue-MRF′ cells. E. coli XL1Blue-MRF′ were grownovernight in either in LB medium (Difco) or NZCYM (Difco) containing 10mM MgSO₄ and 0.2% maltose at 37° C. or 30° C., respectively withshaking. Cells were then centrifuged and resuspended in 0.5 to 1 volumeof 10 mM MgSO₄. 200 μl of this E. coli culture was mixed with severaldilutions of packaged phage library and incubated at 37° C. for 15 min.To this mixture 2.5 ml of LB top agarose or NZCYM top agarose(maintained at 60° C.) was added and plated on LB agar (Difco) or NCZYMagar (Difco) present in 82 mm petri dishes. Phage were allowed topropagate overnight at 37° C. to obtain discrete plaques and the phagetiter was determined.

Screening Genomic Libraries (Plaque Form)

λ Library Plating

E. coli XL1Blue-MRF′ cells were grown overnight in LB medium (25 ml)containing 10 mM MgSO₄ and 0.2% maltose at 37° C., 250 rpm. Cells werethen centrifuged (2200×g for 10 min) and resuspended in 0.5 volumes of10 mM MgSO₄. 500 μl of this E. coli culture was mixed with a phagesuspension containing 25,000 amplified lambda phage particles andincubated at 37° C. for 15 min. To this mixture 6.5 ml of NZCYM topagarose (maintained at 60° C.) was added and plated on 80–100 ml NCZYMagar present in a 150 mm petri dish. Phage were allowed to propagateovernight at 37° C. to obtain discrete plaques. After overnight growth,plates were stored in a refrigerator for 1–2 hrs before plaque liftswere performed.

Plaque Lift

Magna Lift™ nylon membranes (Micron Separations, Inc., Westborough,Mass.) were placed on the agar surface in complete contact with plaquesand transfer of plaques to nylon membranes was allowed to proceed for 5min at room temperature (RT). After plaque transfer the membrane wasplaced on 2 sheets of Whatman 3M™ (Whatman, Hillsboro, Oreg.) filterpaper saturated with a 0.5 N NaOH, 1.0 M NaCl solution and left for 10min at room temperature (RT) to denature DNA. Excess denaturing solutionwas removed by blotting briefly on dry Whatman 3M™ paper. Membranes werethen transferred to 2 sheets of Whatman 3M™ paper saturated with 0.5 MTris-HCl (pH 8.0), 1.5 M NaCl and left for 5 min to neutralize.Membranes were then washed for 5 min in 200–500 ml of 2×SSC, dried byair and baked for 30–40 min at 80° C. The membranes were then probedwith labeled DNA.

DNA Hybridizations

Membranes were prewashed with a 200–500 ml solution of 5×SSC, 0.5% SDS,1 mM EDTA (pH 8.0) for 1–2 hr at 42° C. with shaking (60 rpm) to removebacterial debris from the membranes. The membranes were prehybridizedfor 1–2 hrs at 42° C. (in a volume equivalent to 0.125–0.25 ml/cm² ofmembrane) with ECL Gold™ buffer (Amersham) containing 0.5 M NaCl and 5%blocking reagent. DNA fragments used as probes were purified fromagarose gel using a QIAEX II™ gel extraction kit (Qiagen Inc.,Chatsworth, Calif.) according to manufacturer's protocol and labeledusing an Amersham ECL™ direct nucleic acid labeling kit (Amersham).Labeled DNA (5–10 ng/ml hybridization solution) was added to theprehybridized membranes and the hybridization was allowed to proceedovernight. The following day membranes were washed with shaking (60 rpm)twice at 42° C. for 20 min each time in (in a volume equivalent to 2ml/cm² of membrane) a buffer containing either 0.1 (high stringency) or0.5 (low stringency)×SSC, 0.4% SDS and 360 g/l urea. This was followedby two 5 min washes at room temperature in (in a volume equivalent to 2ml/cm² of membrane) 2×SSC. signals were generated using the ECL™ nucleicacid detection reagent and detected using Hyperfilm ECL™ (Amersham).

Agar plugs that contained plaques corresponding to positive signals onthe X-ray film were taken from the master plates using the broad-end ofa sterile Pasteur pipette. Plaques were selected by aligning the plateswith the x-ray film. At this stage, multiple plaques were generallytaken. Phage particles were eluted from the agar plugs by soaking in 1ml SM buffer (Sambrook et al., (1989) (Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)overnight. The phage eluate was then diluted and plated with freshlygrown E. coli XLlBlue-MRF′ cells to obtain 100–500 plaques per 85 mmNCZYM agar plate. Plaques were transferred to Magna Lift nylon membranesas before and probed again using the same probe. Single well-isolatedplaques corresponding to signals on X-ray film were picked by removingagar plugs and eluting the phage by soaking overnight in 0.5 ml SMbuffer.

Conversion of λ Clones to Plamid Form

To convert the λ ZAP Express™ vector to plasmid form, E. coli strainsXL1Blue-MRF′ and XLOR were used. The conversion was performed accordingto the manufacturer's (Stratagene) protocols for single-plaque excision.

Generation of Probe for Library

Primer Selection

The probe for the library was prepared from a polymerase chain reaction(PCR) fragment of the C. tropicalis ATCC 20336 genome known to representthe FAO gene or genes. By comparing regions of homology between twopublished C. cloacae FAO genes and a published C. tropicalis (NCYC 470)FAOT gene (4), two non-degenerative primers were developed for the PCR:two forward primers designated as (FAO-F1) and (FAO-F2) and one reverseprimer designated as (FAO-R1) (See Table I). These primers wereanticipated to amplify a region in the gene in the open-reading frame(ORF) of the FAO gene.

TABLE 1 Comparison of Primer Sequence to Corresponding Sequence inCandida cloacae. Gene Sequence Comparison Primer C. t. FAOT 352  5′ TCGTGG CGT GAC TCT CCT 3′ 369 (SEQ ID NO:35) FAO-F1 C. c. FAO1 340  5′ TCATGG AGA GAC TCT CCT 3′ 357 (SEQ ID NO:36) C. c. FAO2 340  5′ TCA TGG AGAGAC TCT CCA 3′ 357 (SEQ ID NO:37) C. t. FAOT 608  5′ CTG GTG CTG GTG TAGT 3′ 623 (SEQ ID NO:38) FAO-F2 C. c. FAO1 593  5′ CAG GAG CAG GTG TGG T3′ 608 (SEQ ID NO:39) C. c. FAO2 593  5′ CGG GAG CAG GAG TGG T 3′ 608(SEQ ID NO:40) C. t. FAOT 1780 5′ TTG GTA CCC ATG CTT GTG G 3′ 1762 (SEQID NO:41) FAO-R1 C. c. FAO1 1762 5′ TTG GTA CCC AAG CTT GTG G 3′ 1744(SEQ ID NO:42) C. c. FAO2 1762 5′ TTG GTA CCC AAG CTT GTA G 3′ 1744 (SEQID NO:43)PCR Conditions

The primers selected were used in a PCR reaction with the followingconditions: 5 μl 10×PCR buffer, 5 μl either FAO-F1 or FAO-F2 primerstock (20 μM), 5 μl 30R1 primer stock (20 μM), 1 μl nucleotide mix, 0.5μl Taq polymerase, and 1 μL genomic DNA from Candida tropicalis strainATCC 20336, all in a 50 μl reaction volume. The reaction conditionswere: 95° C. for 2 min, followed by 35 cycles of 95° C. for 30 sec, 53°C. for 30 sec and 72° C. for 1 min. The primer pair, FAO-F1 and FAO-R1,which should have yielded an expected 1429 bp fragment, never yielded aPCR product. The primer pair, FAO-F2 and FAO-R1, which should haveyielded an expected 1173 bp fragment, generated a fragment approximately1200 bp in length.

TOPO TA Cloning of PCR Product

The PCR product was cloned using a TOPO TA cloning kit (Invitrogen,Carlsbad, Ca., TOPO TA Cloning Kit, Version K2, #25-0184) into Top10F′strain cells. The PCR product was first purified by electrophoresis inlow-melt agarose (1%) followed by excising the appropriate band. The gelslice was placed in a microfuge tube and incubated at 65° C. until thegel melted, after which it was placed at 37° C. In a fresh tube 4 μl ofthe melted agarose containing the PCR product was combined with 1 μlTOPO TA cloning vector and was incubated at 37° C. for 10 minutes.Transformation was accomplished by mixing 2–4 μl of this reaction mixwith one vial (50 μl) of E. coli competent cells provided with the kit.These were incubated on ice for 30 min. The cells were heat-shocked at42° C. for 30 seconds without shaking. The vial was immediatelytransferred to ice and 250 μl of room temperature SOC medium was added.This tube was then shaken horizontally at 37° C. for 30 min. Aliquotswere spread on LB plates+100 μg/ml ampicillin upon which 25 μlisopropylthiogalactoside (IPTG, 100 mM)+40 μl Xgal(5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, 40 mg/ml) had beenspread at least 30 minutes prior. Transformants containing inserts weredetermined by blue/white selection, with blue indicating a successfultransformation containing an insert.

White colonies that were positive for the presence of an insert wereinoculated in LB media containing 100 μg/ml ampicillin and grownovernight. Plasmid DNA was obtained from these cultures using theQiaprep kit method (Qiagen, Qiaprep Spin Miniprep Kit #27106), andanalyzed for the presence of the insert by restriction with EcoRI. Inthe PCR 2.1 vector, the insert is flanked on either side by EcoRI sites,so that cutting with EcoRI will release whatever has been inserted intothe plasmid. Several clones showed the correct insert size of about 1200bp. Two of the clones were sequenced and the DNA sequence was comparedto the published sequence for FAOT (12). Since the degree of homologyfor both clones was very high (79% matching bases 608–1199 of the FAOTORF), one of them was selected to prepare the probe DNA.

Preparation of Probe DNA

Several micrograms of plasmid DNA were obtained from one positive cloneand the DNA was digested with EcoRI to release the insert.Electrophoresis of the digest on a 1.2% low-melting agarose gel allowedseparation of the FAO fragment from the PCR 2.1 vector. Theappropriately sized DNA was extracted from the gel (Qiaprep, QIAquickGel Extraction Kit, #28706) and was quantified with a fluorometer. TheFAO DNA fragment was then labeled using the ECL (Amersham, ECL kit, #RPN3001) method.

EXAMPLE 3 Cloning of FAO Genes from Candida tropicalis ATCC 20336

Plates of the λ phage library of C. tropicalis were made and lifts ofthese plates onto nitrocellulose membrane filters were performed,following the procedure described in Materials and Methods (Example 1).Putative positive clones were identified as outlined in Materials andMethods (Example 1). The XLOLR cells containing these library fragmentswere grown up and plasmid DNA was obtained using the Qiaprep kit.Restriction digest and PCR analyses confirmed the presence of an FAOgene in these C. tropicalis library clones. It was known from thesequence information of the probe DNA that at least some of the clonesshould cut with PvuII and KpnI. Therefore, the library clones weredigested with EcoRI, PvuII, and KpnI in single digests, and with PvuIIand KpnI in a double digest. This allowed the direction of the FAO geneto be determined and its placement within the insert of the PBK-CMVvector to be estimated. The initial primers used in preparing the probeDNA, FAO-F2 and FAO-R1, were used to PCR-screen the library clones usingpurified plasmid DNA of these clones as the template. C. tropicalis ATCC20336 genomic DNA was used as the template for the PCR reaction in thecontrol. Eight FAO library clones, designated A1, A4, A5, A6, A8, A9,B5, and B6, were identified as putative positive clones and were sent tothe Sequetech Corporation for sequencing.

When the DNA sequences of the clones were compared to the published FAOTsequence, the clones fell into two groups. Group 1 was composed ofclones A4, A8, B5, and B6. Group 2 was composed of A1, A5, A6, and A9.

4297 bp of the gene from clone A8, which was designated FAO1, wasdouble-strand sequenced (SEQ ID No:1). In addition to the open readingframe (ORF), which was 2112 bp in length, there was 1940 bp upstream and242 bp downstream DNA sequenced. 4158 bp of the gene from clone A9,which was designated FAO2a, was double-strand sequenced (SEQ ID No:3).In addition to the open reading frame (ORF), which was 2112 bp inlength, there was 1520 bp upstream and 523 bp downstream DNA sequenced.There is a CTG codon at bp 2049–2051 in the FAO2 sequence presented inSEQ ID NO:3 (bp 529–531 of the ORF). This CTG is designated as leucinein the universal code, but sequence analysis (14) has confirmed that CTGactually codes for serine in Candida tropicalis ATCC 20336.

Additional sequencing of clone A6 demonstrated close homology to cloneA9. There were a few base pair differences, however, so double strandsequencing of the gene was performed. The results (SEQ ID No:5, Table 2and Table 3) showed that clone A6 was most likely an allele of clone A9.It was designated FAO2b.

The ORF regions of FAO1, FAO2a and FAO2b were compared to analogousregions of the published (12) FAOT gene from Candida tropicalis (NCYC470), the FAO1 and FAO2 genes from Candida cloacae and the FAO gene fromCandida albicans (Table 2).

TABLE 2 DNA Sequence Comparison Between the Subject C. tropicalis FAO1or FAO2 Genes and Published Sequence Data for C. tropicalis NCYC 470, C.cloacae, and C. albicans FAO Genes DNA Sequence #1 DNA Sequence #2 %Identity C. tropicalis FAO1 C. tropicalis FAO2a 82 C. tropicalis FAO1 C.tropicalis FAOT 77 C. tropicalis FAO1 C. albicans FAO 71 C. tropicalisFAO1 C. cloacae FAO1 62 C. tropicalis FAO2a C. tropicalis FAOT 78 C.tropicalis FAO2a C. albicans FAO 73 C. tropicalis FAO2a C. cloacae FAO163 C. tropicalis FAO2b C. tropicalis FAO1 81 C. tropicalis FAO2b C.tropicalis FAO2a 95 C. tropicalis FAO2b C. tropicalis FAOT 77 C.tropicalis FAO2b C. albicans FAO 73 C. tropicalis FAO2b C. cloacae FAO162 C. tropicalis FAOT C. cloacae FAO1 62 C. tropicalis FAOT C. albicansFAO 73 C. cloacae FAO1 C. albicans FAO 59 C. cloacae FAO1 C. cloacaeFAO2 79

The amino acid sequences, which were derived using the universal code,were also compared (Table 3).

TABLE 3 Derived Amino Acid Sequence Comparison Between the Subject C.tropicalis FAO1 or FAO2 Genes and Published Sequence Data for C.tropicalis NCYC 470, C. cloacae, and C. albicans FAO Genes % Iden- %Simi- Protein #1 Protein #2 tity larity Cognis' FAO1 Cognis' FAO2a 81 92Cognis' FAO1 C. tropicalis FAO 82 90 Cognis' FAO1 C. albicans FAO 74 88Cognis' FAO1 C. cloacae FAO1 60 76 Cognis' FAO2a C. tropicalis FAO 85 93Cognis' FAO2a C. albicans FAO 78 88 Cognis' FAO2a C. cloacae FAO1 59 76Cognis' FAO2b Cognis' FAO1 80 91 Cognis' FAO2b Cognis' FAO2a 97 98Cognis' FAO2b C. tropicalis FAOT 85 93 Cognis' FAO2b C. albicans FAO 7688 Cognis' FAO2b C. cloacae FAO1 59 75 C. tropicalis FAO C. cloacae FAO160 77 C. tropicalis FAO C. albicans FAO 76 87 C. cloacae FAO1 C.albicans FAO 55 72 C. cloacae FAO1 C. cloacae FAO2 76 88

The FAO2a and FAO2b genes are 95% identical by DNA sequence and have 97%identity and 98% similarity by amino acid sequence.

The FAO1 and FAO2a genes of the present invention are 82% identical byDNA sequence (81% for FAO2b) and have 81% identity and 92% similarity byamino acid sequence (80% identity and 91% similarity for FAO2b). Incomparison, the C. cloacae FAO1 and FAO2 genes are 79% identical by DNAsequence and have 76% identity and 88% similarity by amino acidsequence. These results indicate that like C. cloacae, C. tropicalisstrain 20336 has two different fatty alcohol oxidase genes.

Interestingly, Vanhanen et al. (4) identified only one FAO gene in theirC. tropicalis NCYC 470 strain. DNA sequence of FAOT was 77% identical tothe FAO1 and FAO2b genes of the present invention and 78% identical tothe FAO2a gene of the present invention. The amino acid sequencecomparison showed that FAOT had 82% identity and 92% similarity to theFAO1 gene of the present invention and had 85% identity and 93%similarity to the FAO2a and FAO2b genes of the present invention.Although the FAOT gene was most similar to the FAO2a gene of the presentinvention, the dissimilarity was still equivalent to about 49 aminoacids out of 704. These data demonstrate that the published FAOT gene isslightly more similar to either of the FAO genes of the presentinvention than the FAO genes of the present invention are to each other.This data further indicates the likelihood that FAO1, FAO2 and FAOT aredifferent genes, rather than alleles of one another.

Although a 1200 bp PCR fragment from all the clones was obtained usingprimers 30F2 and 30R1, the band was generally quite weak, even afteroptimization of the PCR conditions was performed. When the primers,which were designed from the published FAOT sequence, were aligned withthe FAO1. FAO2a and FAO2b genes of the present invention (Table 4),significant lack of homology, particularly with FAO-F1 and FAO-F2, wasobserved. This also indicates that FAO1. FAO2 and FAOT are differentgenes. Even though published sequence information from Candidatropicalis was used to derive the PCR primers, the degree of sequencevariation made finding a functioning PCR primer pair uncertain.

TABLE 4 Comparison of Primer Sequence to Corresponding Sequence inCandida tropicalis FAO1 and FAO2. Gene Sequence Comparison Primer C.t.FAOT 352  5′ TCG TGG CGT GAC TCT CCT 3′ 369 (SEQ ID NO:35) FAO-F1 C.t.FAO1 352  5′ TCG TGG CGT GAC TC C CCT 3′ 369 (SEQ ID NO:44) C.t. FAO2a352  5′ TC T TGG CGT GA T TC C CC G 3′ 369 (SEQ ID NO:45) C.t. FAO2b352  5′ G C C TGG CGT GA T TC C CC G 3′ 369 (SEQ ID NO:46) C.t. FAOT608  5′ CTG GTG CTG GTG TAG T 3′ 623 (SEQ ID NO:38) FAO-F2 C.t. FAO1608  5′ C C G GTG CTG GTG T C G T 3′ 623 (SEQ ID NO:47) C.t. FAO2a608  5′ C C G GTG CTG GTG T CA T 3′ 623 (SEQ ID NO:48) C.t. FAO2b608  5′ C C G GTG CTG GTG T CA T 3′ 623 (SEQ ID NO:49) C.t. FAOT 17805′ TTG GTA CCC ATG CTT GTG G 3′ 1762 (SEQ ID NO:41) FAO-R1 C.t. FAO11780 5′ TTG G C A CCC ATG G TT G G G G 3′ 1762 (SEQ ID NO:50) C.t. FAO2a1780 5′ TTG G C A CCC ATG GC T GTG G 3′ 1762 (SEQ ID NO:51) C.t. FAO2b1780 5′ TTG G CA CCC ATG CC T GTG G 3′ 1762 (SEQ ID NO:52)

EXAMPLE 4 Sub-Cloning and Expression of FAO1 and FAO2 in E. coli

PCR of FAO1 and FAO2 genes

Since the sequence homology data strongly indicated that FAO1 and FAO2were different genes, the uniqueness of the two genes was investigatedby cloning and expressing FAO1 and FAO2a individually in E. coli todetermine the substrate specificity of the two gene products.

The open reading frames of these two genes were amplified by PCR andcloned (Argonne National Labs, Argonne, Ill.) into the self-replicatingvector pJF118EH (13). This vector, containing either the FAO1 or FAO2gene, was transformed into E. coli. Placing the genes for the FAO1 andFAO2 enzymes into E. coli allowed overexpression of the proteins therebyallowing large quantities of enzymes to be generated in a cleanbackground so that their properties could be more clearly defined.

Initially, only the properties of the enzyme derived from the nativesequence of FAO2a were determined. It is known, however that FAO2a has aCTG codon, which is translated as a serine in C. tropicalis but as aleucine in E. coli. Because of that, an FAO2a construct (designatedFAO2a′) was generated having a TCG codon, which codes for serine in bothC. tropicalis and E. coli, in place of the CTG codon. The properties ofboth FAO2a and FAO2a′ were determined as described in the followingexamples.

The primers used to amplify the coding regions of the FAO1 and FAO2genes by PCR are shown below. The restriction sites (underlined) EcoRIand BamHI were added at the 5′ and 3′ ends, respectively. The ATGinitiation codon in the forward primer and the dual termination codonsin the reverse primers are shown in itialics.

FAO1U 5′-CCGAATTCGACATGGCTCCATTTTTG-3′ (SEQ ID NO:53) FAO1L5′-CCGGATCCATTACTACAACTTGGCCTTGGT-3′ (SEQ ID NO:54) FAO2U5′-CCAGTGAATTCAGATGAATACCTTCT-3′ (SEQ ID NO:55) FAO2L5′-CCGGATCCCCGTCTCACTACAACTTG-3′ (SEQ ID NO:56)PCR used Platinum Pfx DNA Polymerase from Life Technologies, Inc.(Rockville, Md. 20849-6482). The reaction conditions for each 50 μlreaction were:

-   -   1× buffer (supplied by manufacturer)    -   1.0, 1.5 or 2.0 mM MgSO₄    -   1 μM each primer    -   0.2 mM each of the 4 dNTPs    -   200–400 ng FAO1 or FAO2 plasmid    -   1 unit Platinmum Pfx Polymerase

Reactions were incubated in a Robocycler Gradient 96 thermocycler(Stratagene) for one cycle at 94° C. for 2 minutes, followed by 30cycles at 94° C. (30 seconds); 55° C., 58° C. or 61° C. (45 seconds);72° C. (2 minutes). The reactions were completed by incubation at 72° C.(10 minutes) for 1 cycle.

FAO1 gave the expected product (2.1 kb) under all of the conditionstested. The optimum conditions for FAO2 were at 1.0–1.5 mM MgSO₄ and 55°C.–58° C.

Agarose Gel Purification of PCR Products

Three of the PCR reactions for each gene were pooled and purified withthe QIAquick-spin PCR Purification Kit (Qiagen) following themanufacturer's instructions. The DNA was then fractionated on a 1.0%agarose gel. The 2.1 kb bands were removed and the DNA extracted withthe QIAEX II Gel Extraction Kit (Qiagen) following the manufacturer'sinstructions.

Ligation into pJF118EH

The expression vector pJF118EH (13) was digested with EcoRI and BamHI,and fractionated on a 1.0% agarose gel. The band was excised andpurified with the QIAEX II Gel Extraction Kit (Qiagen). The FAO1 andFAO2 PCR products were digested with EcoRI and BamHI, and gel purifiedin the same manner. The digested bands were visualized on an agarose gelbut the exact concentrations were not determined.

Ligation reactions (20 μl) containing 1 μl of FAO1 or FAO 2 and 4 μlpJF118EH in 1× ligation buffer (Promega) with 1 μl (3 units) T4 DNAligase (Promega) were incubated for 2 hours at 25° C. 100 μl of LibraryEfficiency DH5 E. coli (Life Technologies, Inc.) were transformed with1.5 μl of each ligation reaction.

Six colonies from each transformation were selected, miniprep DNA wasprepared and insert size determined by digestion with EcoRI and BamHI. 5of the 6 FAO1 clones contained the 2.1 kb insert. All 6 of the FAO2clones contained the 2.1 kb insert.

One clone of each was selected and glycerol stocks were stored at −80°C. Samples of each clone were also used for expression and enzymeactivity analysis. The FAO1 clone, designated FAO1-EC, contained plasmidFAO1jf (see FIG. 1), and the FAO2 clone, designated FAO2-EC, containedplasmid FAO2jf (see FIG. 2). Both FAO genes in these plasmids weresequence confirmed by Sequetech Corporation, Mountain View, Calif.

Induction of FAO1 and FAO2

Overnight cultures of FAO1-EC and FAO2-EC were grown at 30° C. in 5 mlof Terrific Broth (TB) (Sigma Chemical Co, St. Louis, Mo.) plus 100μg/ml ampicillin at 250 rpm. 50 ml of TB plus 100 μg/ml ampicillin wasplaced in each of two 500 ml baffled flasks. TB plus 100 μg/mlampicillin was used for both the starter cultures and the cultures thatwere induced to produce the enzyme. The flasks were inoculated with theovernight cultures to an absorbance at 600 nm of approximately 0.2. Thecultures were grown at 30° C., with shaking at 250 rpm. When eachculture had reached an absorbance at 600 nm of 5 to 6, it was inducedwith IPTG to a final concentration in each culture of 1 mM. The cultureswere then allowed to incubate another three hours post-induction. Thecells were harvested by centrifugation (Sorvall RC5C) at approximately6,000×g (GS3 rotor at 6,000 rpm) for 10 minutes. The supernatant spentbroth (SB) was removed and saved, and the cell pellets were frozen at−20° C. for later use.

Preparation of E. coli Cell Extracts

Microsomes were prepared for alcohol oxidase assays by resuspending theinduced cell pellets in 50 ml PO₄/glycerol buffer. 500 μl of a 100 mMPMSF solution was added for a final concentration of 1 mM of PMSF in thesuspension. The cells were broken by passage twice through a chilledFrench pressure cell, and were examined by phase contrast microscope toassure that 95% or greater of the cells had been broken. The broken cellsuspension (BCS) was centrifuged at approximately 37,000×g for 30minutes to pellet the cellular debris. The supernatant (cell freeextract, CFE) was removed and saved for assays. The pellet of cellulardebris (CD) was resuspended in 50 ml of phosphate-glycerol buffer sothat it was equal to the original concentration of the cells in theculture.

Preparation of E. coli Microsomal Suspensions

The cell-free extracts were warmed to room temperature and thoroughlymixed before a 3 ml sample of each was removed for the microsomalpreparation. These samples were centrifuged in an ultracentrifuge at100,000×g for one hour at 4° C. to pellet the microsomes. Thesupernatant was then removed and saved, and each microsomal pellet wasresuspended in 0.5 ml PO₄/glycerol buffer for a 6× concentration of theFAO enzyme.

EXAMPLE 5

Construction of a CTG-Codon-Altered FAO2a Gene

A codon alteration of FAO2a was performed by overlap-extension PCR. Thefollowing primer sets were used to generate the initial constructs:

A9.1N 5′ ATC AAC GCC ACC CCA ACC 3′ (SEQ ID NO:57) FAO2-CTG-R 5′ GGT TTCTCC ATA AAC GAG TAC CTG AAAGGG TCA ACC 3′ (SEQ ID NO:58)

These primers were designed to cover the region 208 bp 3′ to 545 bp 3′of the start of the ORF yielding a fragment of 338 bp in length. The CTGcodon alteration to CGA (TCG in reversed complement) is indicated inbold. A former KpnI restriction site (GGTACC), underlined above, hasbeen eliminated by this codon alteration.

PCR used Platinum Pfx DNA Polymerase from Life Technologies, Inc. Thereaction conditions for each 50 μl reaction were:

-   -   1× buffer (supplied by manufacturer)    -   1.0 mM MgSO₄    -   1 μM each primer    -   0.3 mM each of the 4 dNTPs    -   200–400 ng FAO2a plasmid    -   1 unit Platinmum Pfx Polymerase

A9.1E 5′ ATC TGT CTA GCA AAG GTC 3′ (SEQ ID NO:59) FAO2-CTG-F 5′ GGT TGACCC TTT CAG GTA CT C GTT TAT GGA GAA ACC 3′ (SEQ ID NO:60)

These primers were designed to cover the region 510 bp 3′ to 2069 bp 3′of the start of the ORF yielding a fragment of 1560 bp in length. TheFAO2-CTG-F and FAO2-CTG-R are overlapping primers.

This PCR also used Platinum Pfx DNA Polymerase from Life Technologies,Inc. The reaction conditions for each 50 μl reaction were:

-   -   1× buffer (supplied by manufacturer)    -   5.0 mM MgSO₄    -   1 μM each primer    -   0.3 mM each of the 4 dNTPs    -   200–400 ng FAO2a plasmid    -   1 unit Platinmum Pfx Polymerase

Reactions were incubated in a PTC200 thermal cycler (MJ Research) forone cycle at 94° C. for 2 minutes, followed by 30 cycles at 94° C. (15seconds); 55° C. (30 seconds); 68° C. (4 minutes). The reactions werecompleted by incubation at 72° C. (7 minutes) for 1 cycle.

The appropriate-sized PCR fragments were separated on 1% low-meltingagarose gel, and were then excised into separate microfuge tubes. Athird PCR was performed using primers A9.1N and A9.1E. This PCR alsoused Platinum Pfx DNA Polymerase from Life Technologies, Inc. Thereaction conditions for each 50 μl reaction were:

-   -   1× buffer (supplied by manufacturer)    -   2.0 mM MgSO₄    -   1 μM each primer    -   0.3 mM each of the 4 dNTPs    -   1 μl each PCR fragment (338 bp and 1560 bp, low melt agarose        melted at 65° C.)    -   1 unit Platinum Pfx Polymerase

The reaction was incubated in a PTC200 thermal cycler (MJ Research) forone cycle at 94° C. for 2 minutes, followed by 30 cycles at 94° C. (15seconds); 60° C. (30 seconds); 68° C. (2 minutes). The reactions werecompleted by incubation at 72° C. (7 minutes) for 1 cycle. This resultedin a PCR fragment that was 1862 bp in length and covered from 208 bp to2069 bp from the start of the ORF. This fragment was TOPO-TA cloned andthe resulting plasmid was prepared as described in Materials and Methods(Example 1).

In order to replace the CTG codon in plasmid FAO2jf with the modifiedsequence, the plasmid, FAO2jf, was cut with KpnI and MfeI to remove afragment 470 bp in length, leaving the major portion of the plasmid,6924 bp intact. The 1862 bp fragment was also cut with KpnI and MfeI toremove a fragment 470 bp in length. The appropriate fragments, the 6924bp fragment from plasmid FAO2jf and the 470 bp fragment cut from the1862 bp fragment containing the modified CTG codon, were purified on 1%agarose gel, and then were extracted from the gel using Qiagen'sQiaquick kit.

After obtaining the appropriate DNA fragments, a ligation was set upusing the Quick Ligation product and protocol from New England Biolabs.This ligation was transformed back into DH5α cells. Plasmid preparationsfrom putative clones were screened by cutting with KpnI and Mfe I. Oneof the positive clones was selected for further study and was designatedFAO2a′EC. Since all of the CTG-modified FAO2a gene (designated FAO2a′)except for the 469 bp fragment containing the codon modification hadbeen previously sequence verified, only this 469 bp portion of the genewas sequence-verified. The gene did have the appropriate codon changedfrom a CTG to a TCG (bp 2049–2051 in SEQ ID NO:9. It also had a secondmutation, presumably generated by PCR. The mutation was at bp 2117 andresulted in a codon change from ATT to ATC, both of which code forisoleucine.

Amplification Cassette Preparation for FAO1 and FAO2

The initial step in creation of the amplification cassette of FAO1 andFAO2 was to use primers with PacI restriction sites on the ends toamplify the ORF and upstream regions of each gene. For FAO1 the primersused were: (PacI restriction site underlined)

FAO1-F3-PacI 5′ CCT TAA TTA ATG CAT ACT CGG AGC ATA TCG C 3′ (SEQ IDNO:61) FAO1-R3-PacI 5′ CCT TAA TTA ATG GGC GGA ATC AAG TGC C 3′ (SEQ IDNO:62)The region covered is 1939 bp 5′ and 245 bp 3′ of the ORFFor FAO2 the Primers were:

FAO2-F1-Pac I 5′ CCT TAA TTA ATC TCA CCA AGT ACG AGA ACG 3′ (SEQ IDNO:63) FAO2-R1-Pac I 5′ CCT TAA TTA AGA CGC AAG CAC AGG TGC C 3′ (SEQ IDNO:64)The region covered is 1520 bp 5′ and 526 bp 3′ of the ORF

The Accutaq LA Core Kit (Sigma, #ACCUCORE) was used to create the PCRfragments. This kit contains both a Taq DNA polymerase with a smallamount of a proofreading enzyme. The reaction mixture was preparedfollowing the recommendations in the kit. The PCR conditions followedwere: 98° C. for 30 see followed by 35 cycles of: 94° C. for 5 sec, 65°C. for 20 see and 68° C. for 5 min. Following this there was a finalextension of 72° C. for 5 min. These PCR fragments were TA-TOPO clonedinto PCR 2.1 vector, which was transformed into E. coli strain Top10F′cells, creating the strains FAO1:PacI and FAO2:PacI.

In order to replace most of the ORF sequence obtained by PCR withgenomic DNA, FAO1:PacI was cut with AatII and NheI to release a fragmentapproximately 1.9 kb in length. Its complementary library clone, A8.1,was also cut with AatII and NheI. FAO2:PacI and its complementarylibrary clone, A9.1, were cut with NheI and BstBI to release a 3.2 kbfragment. The appropriate fragments were purified on 1% low-meltingagarose gel, and then were extracted from the gel using Qiagen'sQiaquick kit.

After obtaining the appropriate DNA fragments, a ligation was set up forboth FAO1 and FAO2 in which the ORF from the library clones was ligatedinto the PCR 2.1 vector with the upstream and downstream regions of thegene. These ligations were transformed back into Top10F′ cells. Plasmidpreparations from putative clones were screened by cutting with AatIIand NheI for the FAO1 clones, or cutting with NheI and BstBI for theFAO2 clones, and cutting with PacI for all clones.

Because the beginning and end of each ORF was obtained from the originalPCR amplification of each gene, these areas needed to be sequenced tomake sure that no mutations had been introduced into the ORF. ThreeFAO1:PacI ORF replacement clones were sent to Sequetech Corp. (MountainView Calif.) for sequencing. Five FAO2:PacI ORF replacement clones werealso sent to Sequetech Corp. for sequencing. The FAO1: PacI ORFreplacement clones 1 and 2, and FAO2:PacI ORF replacement clones 1–5were all mutation free, and could be used for making amplificationcassettes.

EXAMPLE 6 Preparation of a Promoter Fusion Construct

Fusions between the promoter of the cytochrome p450 monooxygenase geneCYP52A2, and the ORF of either FAO1 or FAO2 were prepared by overlapextension PCR. PCR reactions for both the CYP52A2 promoter and FAO ORFwere set up using the Accutaq PCR kit. The primers used to obtain theCYP52A2 promoter for the fusion with FAO1 were (PacI restriction site):

00218-179A 5′ CCT TAA TTA AAG TCT CCA AGT TGA CCG AC 3′ (SEQ ID NO:65)FAO1-A2-R 5′ AAA TGG AGC CAT GGT CGT GAT GTG TG 3′ (SEQ ID NO:66)

FAO-A2-R was designed to sit at the 3′ end of the CYP52A2 promoter, butthe last half of the 5′ to 3′ end of the primer was derived from thecomplementary sequence of the beginning of the FAO1 ORF. Conversely, theprimers used to amplify the FAO1 ORF were:

FAO1-A2-F 5′ CAC ATC ACG ACC ATG GCT CCA TTT TTG CC 3′ (SEQ ID NO:67)FAO1-R3-Pac I 5′ CCT TAA TTA ATG GGC GGA ATC AAG TGC C 3′ (SEQ ID NO:68)

FAO1-A2-F was designed to sit at the 5′ end of the FAO1 ORF, with thefirst half of the 5′ to 3′ end of the primer derived from thecomplementary sequence of the end of the CYP52A2 promoter. In this way,the CYP52A2 promoter and FAO ORF regions produced had complementarysequences to one another.

The primers used to obtain the CYP52A2 promoter were:

00218-179A 5′ CCT TAA TTA AAG TCT CCA AGT TGA CCG AC 3′ (SEQ ID NO:69)FAO2-A2-R 5′ GAA GGT ATT CAT GGT CGT GAT GTG TG 3′ (SEQ ID NO:70)The primers used for the FAO2 ORF regions were:

FAO2-A2-F 5′ CAC ATC ACG ACC ATG AAT ACC TTC TTG CC 3′ (SEQ ID NO:71)FAO2-R1-Pac I 5′ CCT TAA TTA AGA CGC AAG CAC AGG TGC C 3′ (SEQ ID NO:72)

Once the appropriate PCR bands had been obtained, each PCR fragment waspurified on a 1% low-melt agarose gel, which was then cut from the gel,and the PCR product extracted. The CYP52A2 promoter band wasapproximately 1.1 kb for both fusion reactions. The ORF band for FAO1was approximately 2.4 kb and the ORF band for FAO2 was approximately 2.7kb. The fusion PCR was set up using a small portion of the CYP52A2promoter and FAO ORF gel-purified bands as template DNA. The Accutaq kitwas used, and the primers were the forward primer for the CYP52A2promoter and the reverse primer for each FAO ORF. During the PCRreaction, the CYP52A2 promoter and FAO ORF annealed together at theircomplementary ends, and the PCR reaction continued over the junction toform the fusion construct. The _(pCYP52A2)FAO1 fusion band was 3.5 kband the _(pCYP52A2)FAO2^(a) fusion band was 3.8 kb.

These fusion bands were then TA-TOPO cloned and transformed into Top10F′E. coli cells. Once again, the majority of the ORF frame of each fusionclone was replaced with corresponding genomic DNA because of thepossibility of the PCR reaction introducing mutations into the ORF. The_(pCYP52A2)FAO1 clones were cut with the restriction enzymes AatII andNheI to remove most of the FAO1 ORF. The _(pCYP52A2)FAO2^(a) clones werecut with the restriction enzymes AatII and BstBI. A correspondinggenomic fragment for each FAO ORF was obtained in a similar manner.After digesting, the appropriate fragments of each gene were purifiedand ligated together. The resulting plasmid contained the _(pCYP52A2)FAOfragment with the replaced FAO genomic DNA in the PCR 2.1 vector. Thiswas transformed into Top10F′ cells and three of the _(pCYP52A2)FAO1 andfive of the _(pCYP52A2)FAO2^(a) clones were sent to SequetechCorporation for sequencing.

After sequencing, all three of the _(pCYP52A2)FAO1 ORF replacementclones were shown to have no mutations and were suitable for makingamplification cassettes. Sequencing showed that all five of the_(pCYP52A2)FAO2^(a) ORF replacement clones had apparent mutationsintroduced by the PCR in various places in the ORF.

Creation of amplification cassettes for _(pCYP52A2)FAO1 fusion clone wasaccomplished by removing the fragment from the PCR 2.1 vector andcloning it into the pURA3in vector. This vector consisted of pNEB193with inverted fragments of the URA3 gene from C. tropicalis flankingeither region of the insertion site. The insertion site is a single PacIrestriction enzyme site, into which the _(pCYP52A2)FAO1 fusion clone canbe inserted after restriction digest with PacI and gel purification. TheURA3 gene fragments are flanked by AscI and PmeI restriction digestsites so the entire fragment of the FAO clone insertion flanked by theURA3 inverted gene sequences can be removed and used to transform aCandida tropicalis ura⁻ base strain. This was successfully performedusing _(pCYP52A2)FAO1.

EXAMPLE 7 Results

Alcohol Oxidase Activity in Fermentation Samples

During the course of a diacid fermentation with strain HDC23-3 usingHOSFFA as substrate, FAO activity generally did not appear untilapproximately 2–4 hours post-induction with HOSFFA (see FIG. 3).Activity rose rapidly to peak at approximately 30–40 hourspost-induction, and then dropped approximately 5 to 10 fold by 100–120hours post-induction. Although this trend remained consistent among mostof the fermentation runs, the time and height of the peak of activitydid vary somewhat.

One of the difficulties encountered in measuring FAO activity in theextracts was the presence of catalase in the cells. Catalase convertshydrogen peroxide in the cell into water and molecular oxygen. Catalasein the extracts could compete with the horseradish peroxidase for thehydrogen peroxide produced by the oxidation of the alcohol to thealdehyde by the FAO, which could result in activity measurements thatwere lower than the actual activity. No FAO activity was found in themicrosomal supernatant, but there was activity in the resuspendedmicrosomal pellet, and this activity was approximately the same as thatin the original extract. Catalase activity was present in the microsomalpellet, but at approximately 1/100 the level found in the originalextract. This data indicates that, under the assay conditions used, thecatalase was not significantly competing with the horseradish peroxidasefor hydrogen peroxide in the reaction mixture.

EXAMPLE 8 Fractionation of Alcohol Oxidase Activity

E. coli cells containing the plasmids, FAO1jf or FAO2jf, were induced toexpress FAO1 or FAO2 as described in Materials and Methods (Example 1).The spent broth, cell free extract, and the resuspension of the cellulardebris (see Materials and Methods, Example 1) were all assayed for FAOactivity in an effort to determine the location of the FAO activity. Ifdeposited extracellularly, FAO activity would be seen in the spentbroth. If deposited internally, FAO activity would be detected in thecell free extract or in the resuspended cellular debris. Soluble enzymeor membrane-bound microsomal enzyme would be found in the cell freeextract. To determine the cellular location of the alcohol oxidase,activity assays were performed with the various fractions. No FAOactivity was seen in the spent broth, and the activity found in theresuspension of the cellular debris pellet (FAO1=0.002 U/ml; FAO2=0.002U/ml) was one-fifth to one-tenth the activity determined for cell freeextract (FAO1=0.026 U/ml; FAO2=0.044 U/ml).

Microsomal preparations were made from the cell-free extracts of theseFAO1 and FAO2 E. coli cultures. Activity in each resuspended microsomalpellet and supernatant was tested for FAO activity. No activity was seenin the supernatant from the FAO1 microsomal pellet, and the activity inthe supernatant from the FAO2 microsomal pellet (0.010 U/ml) wasapproximately one-fifth of the activity in the resuspended FAO2microsomal pellet (0.050 U/ml). The activity in the FAO2 microsomalpreparation was almost twice the activity that was seen in the FAO1microsomal preparation (0.031 U/ml). These data indicate that themajority of the enzymatic activity was contained in the microsomesisolated from the E. coli cultures.

Optimization of Induction

In order to determine the optimal temperature for synthesis of the FAO1and FAO2a enzymes in E. coli cultures, seed cultures were grown upovernight at 37° C. and 250 rpm in Terrific broth. 50 ml of Terrificbroth was added to each of four 500 ml baffled flasks; one each for FAO1and FAO2a to be incubated at either 30° C. or 37° C. Each flask wasinnoculated with the overnight cultures to an absorbance at 600 nm ofbetween 0.7 and 0.8, and was then incubated with shaking at 250 rpm.When each culture had reached an absorbance at 600 nm of approximately4.5, it was induced with IPTG to a final concentration of 1 mM. Thecells were harvested at 3 hours post-induction by centrifugation at6000×g for 10 minutes. The supernatant was removed and the cells werestored at −20° C. The FAO1 and FAO2a E. coli cultures grown at 30° C.and 37° C. were prepared for enzyme assays as described in Materials andMethods. The cell free extract prepared from each of these cultures wasassayed for FAO activity. It was found that growth at 30° C. resulted ina greater production of FAO1 (14.7 U/ml) and FAO2 (2.44 U/ml) thangrowth at 37° C. (8.8 U/ml for FAO1 and 1.33 U/ml for FAO2a).

In a second set of experiments, the optimal concentration of theinducer, IPTG, was determined for E. coli containing either FAO1 orFAO2a. The growth experiment was set up in the same way as the previousexperiment, with overnight seed cultures of FAO1 and FAO2a grown up at37° C. in Terrific Broth plus 100 μg/ml ampicillin. 50 ml Terrific brothwas added to each of six flasks; three flasks each for FAO1 and FAO2a.One flask from each set was induced with either 0.5 mM IPTG, 1.0 mMIPTG, or 2.0 mM IPTG. The cultures were grown at 30° C. with shaking at250 rpm. When each culture had reached an absorbance at 600 nm ofapproximately 5.0, it was induced with the appropriate concentration ofIPTG. The cultures were allowed to grow another 2.5 hours post-inductionbefore harvesting the cells by spinning at 6000×g for 10 minutes. Thecell pellets were stored at −20° C. Cell free extracts were prepared andafter assaying for alcohol oxidase activity, it was found that there waslittle dependence of the FAO1 activity on IPTG concentration (0.5mM=13.24 U/ml; 1 mM=13.89 U/ml; 2 mM=14.3 U/ml). A similar response toIPTG concentration was observed for FAO2a (0.5 mM=4.74 U/ml; 1 mM=5.68U/ml; 2 mM=4.65 U/ml). A standard concentration of 1 mM IPTG was chosenfor induction of FAO1, FAO2a and FAO2a′.

TABLE 5 Alcohols Used for Substrate Specificity Testing ActivityActivity Detected* Compound Tested Detected Compound Tested 2-Pentanol1-Phenylpropan-1-ol 2-Hexanol 3-Phenylpropan-1-ol X 2-Decanol2-Phenylbutan-1-ol X 2-Undecanol Methanol X 2-Dodecanol Ethanol X2-Hexadecanol Propanol 3-Octanol Butanol X 10-Undecen-1-ol X Hexanol1,8-Octanediol X Octanol X 1,2-Octanediol X Decanol X 1,10-Decanediol XDodecanol X 1,2-Dodecanediol X Tetradecanol X 1,16-Hexadecanediol XHexadecanol X 10-Hydroxydecanoic acid 4-cyclohexyl-1-butanol X12-Hydroxydodecanoic 3-cyclohexyl-1- acid propanol X16-Hydroxydodecanoic 2-cyclohexylethanol acid CitronellolCyclohexylmethanol Geraniol Linalool *“X”= Activity detected using thissubstrate with either FAO1 or FAO2Substrate Specificity of FAO1, FAO2a and FAO2a′

FAO1, FAO2a and FAO2a′ were tested for their activity with variousalcohols. The alcohols shown in Table 5 were prepared in 20 mM stocksolutions in acetone. Alcohols showing activity with either FAO1 orFAO2a are indicated. The final concentration of the alcohol used in thesubstrate specificity experiment was 200 μM. These same alcohols wereused to determine the Km and Vmax of FAO1, FAO2a, and FAO2a′. Thesubstrate specificity profiles FAO1, FAO2a, and FAO2a′ were reported aspercentages of activity for dodecanol, with dodecanol arbitrarily set at100% activity. The activity of FAO1, FAO2a, and FAO2a′ with 1-alkanolsis shown in FIG. 4. Interestingly, FAO1 preferred 1-octanol assubstrate, with 1-tetradecanol being the preferred longer-chain alcohol.In contrast, FAO2a and FAO2a′ preferred 1-dodecanol above all other1-alkanols. There was a big drop in activity in going from a C8 alcoholto a C6 alcohol with FAO1, FAO2a, and FAO2a′. The activity of the threeenzymes on 2-alkanols is shown in FIG. 5. FAO2a and FAO2a′ oxidize2-octanols fairly well; however no activity was detected with FAO1. Incontrast, FAO1 oxidizes ω-hydroxy fatty acids well, but no activity forω-hydroxy fatty acids was measured with FAO2a and FAO2a′ (FIG. 6). Theseresults indicate that FAO1 and FAO2a appear to be very differentenzymes, with significant differences in substrate specificity.Interestingly FAO2a and FAO2a′ oxidize 10-undecen-1-ol much better thanFAO1. Within experimental error, the substrate specificity of FAO2a andFAO2a′ are essentially the same, indicating that having a serine or aleucine at amino acid position 177 has little if any effect on thesubstrate specificity of the enzyme. Although the substrate specificityof FAO2b was not performed, due to the close homology with FAO2a, it islikely to be very similar.

Extracts made from fermentor samples induced with HOSFFA show goodactivity with 16-hydroxyhexadecanoic acid, but no activity with2-dodecanol (data not shown). Hence, it appears that FAO1 is induced toa much greater extent than FAO2a and, at least in HOSFFA fermentations,FAO1 appears to be the predominant fatty alcohol oxidase.

Km Determinations

K_(m) values indicate the affinity of an enzyme for the substrate beinginvestigated. K_(m) values for most of the alcohols that demonstratedactivity with FAO1, FAO2a, and FAO2a′ were determined by measuringenzyme activity while keeping the concentration of enzyme constant andvarying the concentration of alcohol added to the reaction mixture.

Most of the K_(m) values were determined at pH 7.6 using theHEPES/Triton X-100 buffer and stock solutions of alcohols, which wereprepared at concentrations of 5 mM and or 1 mM, were dissolved in thissame buffer. For FAO2a′, the Km values were determined in 0.1 M HEPES,pH 7.6, and the stock solutions of substrate were dissolved in acetone.In this case, all reactions contained the same amount of acetone (20μl/ml). The results are shown in Table 6.

TABLE 6 Km Values for FAO1, FAO2a, and FAO2a¹. FAO1 FAO2a FAO2a¹ Corr.Range of Values Corr. Range of Values Corr. Range of Values Substrate KmCoeff. Low High Km Coeff. Low High Km Coeff. Low High Octanol 41.0 μM0.9996 35.0 49.3 503 μM 0.9977 329.3 1069.2 654 μM 0.9976 323.5 30607.9Decanol 15.1 μM 0.9912 11.8 21.3 74 μM 0.9990 64.3 85.9 69.6 μM 0.997865.1 74.8 Dodecanol 14.9 μM 0.9938 11.9 19.9 2.5 μM 0.9907 2.4 2.5 4.4μM 0.9928 3.9 4.4 Tetradecanol 28.1 μM 0.9976 20.8 43.3 4.6 μM 0.99684.5 4.8 2.4 μM 0.9751 2.2 2.5 Hexadecanol 11.9 μM 0.9998 11.1 11.8 56 μM0.9977 51.2 61.7 85 μM 0.9987 78.4 93.5 2-Decanol NR 934 μM 0.9991 823.21078.5 1090 μM 0.9834 567.3 13696.9 2-Undecanol NR 141 μM 0.9970 125.9159.0 162 μM 0.9913 150.9 174.5 2-Dodecanol NR 41.3 μM 0.9990 39.6043.06 75.3 μM 0.9980 67.9 84.5 2-Hexadecanol NR 350 μM 0.9992 332.5370.0 720 μM 0.9982 628.1 844.2 1,2-dodecanediol NR 998 μM 0.9935 720.21622.7 928 μM 0.9969 742.6 1237.3 1,10-decanediol 67.0 μM 0.9997 58.977.6 425 μM 0.9985 394.4 462.3 1607 μM 0.9975 1377.2 1927.51,16-hexadecanediol 10.3 μM 0.9995 9.3 11.6 37.3 μM 0.9969 33.9 41.516.3 μM 0.9978 15.7 16.9 10-undecene-1-ol  9.9 μM 0.9963 8.8 11.3 45 μM0.9986 40.7 50.1 36.1 μM 0.9916 32.7 40.4 12-hydroxy-decanoic 99.0 μM0.9930 78.2 134.4 NR NR 16-hydroxy-  7.4 μM 0.9912 5.9 9.9 NR NRhexadecanoicFor most alcohols tested, FAO1 yielded K_(m) values between 10–20 μM.The lowest Km value was found with 16-hydroxy-hexadecanoic acid. Usingeither FAO2a and FAO2a′ as enzyme, the Km values for the variousalcohols were very similar, again demonstrating that having a serine orleucine at amino acid position 177 does not have much effect on theaffinity of the enzyme for a particular substrate. For FAO2, the Kmvalues were generally much more variable and yielded values ranging from2 μM to greater than 1 mM. The substrates with the lowest Km values were1-dodecanol and 1-tetradecanol. Based on these results, it is logicalthat FAO1 appears to be the predominant fatty alcohol oxidase producedin HOSFFA fermentations, since ω-hydroxy fatty acids are intermediatesin the oxidation of fatty acids to diacids.

EXAMPLE 9

Amplification of FAO1 in Candida tropicalis

In fermentations with HOSFFA as substrate, ω-hydroxy fatty acids areproduced at levels between 0.1% to 0.5% (w/w) in fermentation broth.This indicates that there is a minor bottleneck at the second step indiacid production, i.e. the conversion of the alcohol to the aldehyde.This ω-hydroxy fatty acid has been found to interfere with purificationof the diacid and causes a significant loss in recovery. Therefore, itis important to reduce the level of ω-hydroxy fatty acids produced inthe fermentation. Since the substrate specificity tests and K_(m)determinations performed with FAO1 and FAO2 indicated that FAO1 was thepredominant fatty alcohol oxidase produced during the HOSFFAfermentation, amplification of the FAO1 gene was initially pursued.

Amplification cassettes with PacI restriction sites at the ends of theFAO1 and FAO2 genes were prepared as described in Materials and Methods(Example 1). Neither of these constructs has been tested in Candidatropicalis, however. A construct in which the promoter from the CYP52A2gene replaced the native promoter in FAO1 was successfully prepared (SEQID NO:7) and transformed into Candida tropicalis. The CYP52A2 geneencodes a cytochrome P450 monooxygenase that is part of the hydroxylasecomplex responsible for catalyzing the first step in diacid production,i.e. the conversion of the fatty acid to the alcohol. By fusing thepromoter region of this gene to the ORF of the FAO1 gene, it washypothesized that the alcohol oxidase gene and corresponding enzymewould be induced earlier and more strongly than it might otherwise be.This would make the conversion to the diacid faster and help to reducethe bottleneck at this point in the reaction. Three clones, namedHDC40-1, HDC40-5 and HDC40-7 were selected for testing in fermentors. Bycomparing band intensities in a Southern blot analysis and estimatingcopy numbers, HDC40-1 appears to have a low (one additional copy ofFAO1) copy number; HDC40-5 has a higher copy number (two additionalcopies); and HDC40-7 has multiple additional copies per cell.

These strains were tested in fermentations with HOSFFA as substrate.FIG. 7 shows that, under similar fermentation conditions, all strainsproduced more diacid than the base strain, H5343. Both HDC40-1 andHDC40-7 had similar initial productivity values up to about 24 h ofbioconversion time. HDC40-1, however, maintained a higher level ofproductivity than HDC40-7 over the next 48 h. FIG. 8 shows the level ofω-hydroxy fatty acids produced in HDC40 strains, relative to the basestrain, H5343, which has no amplified genes. Note that, although thereis still some ω-hydroxy fatty acids produced, the levels areconsiderably reduced, compared to H5343. An alcohol oxidase activityprofile (FIG. 9) determined over the course of the fermentationdemonstrates that the alcohol oxidase, as expected, has beenconsiderably amplified and is very active during the early hours of thefermentation. When ricinoleic acid is used as a fermentation substratein place of HOSFFA, high levels of ω-hydroxy fatty acids (relative tothe amount of diacid produced) are detected during the fermentation. Forthis reason, ricinoleic acid fermentations are a more definitive test ofthe effectiveness of the FAO1 amplification. When HDC40-1, HDC40-5 andHDC40-7 fermentations were compared, it was discovered that the FAO1gene copy number inversely correlated with the reduced level ofω-hydroxy fatty acids, i.e. the percent of ω-hydroxy fatty acidsproduced relative to the total level of oxidized product (FIG. 10).

EXAMPLE 10 Identification of Signature Motifs Unique to FAO1 and FAO2

The amino acid sequences corresponding to the subject FAO1 and FAO2genes were examined for the presence of one or more of the seven peptidesequences identified by Slabas et al. (12) as indicative of an FAO gene.Table 7 shows a comparison of the signature peptides identified bySlabas et al. among the FOA1 and FAO2 genes of the present invention,and the FAOT gene of C. tropicalis. Also compared are similar sequencesfrom the Candida albicans FAO enzyme (FAOCA). As reflected in Table 7,all seven FAOT peptides agree with the signature peptides identified bySlabas et al. Six out of the seven FAO1 peptides but only four of theseven FAO2 peptides agree with the signature peptides. Interestingly,although FAO2 is closest to FAOT in amino acid sequence identity andsimilarity (Table 3), FAO1 is most similar to FAOT when the sevensignature peptides are compared.

Peptide 4 of FAO1 and FAO2 (CGFCYLGC) is a unique peptide, not found inpreviously characterized FAOs. Peptide 1 of FAO2a and FAO2b(IIGSGAGAGVMA) as well as peptide 4 of both FAO1 [and] FAO2a and FAO2b(CGFCYLGC) is also a unique peptide, not found in previouslycharacterized FAOs.

TABLE 7 Comparison of Signature Peptides of Cognis' FAO1 and FAO2 withC. tropicalis FAOT and C. albicans FAOCA Peptide 1 Peptide 2 Peptide 3Peptide 4 Peptide 5 Peptide 6 Peptide 7 Signature IIGSG(X)GAGVVAAGSTFGGG NWSACLKTP CG(X)CHLGC IG(X)NL(X)LHPVS SAHQMS(X)CRMSG PTASG(X)NPMPeptide^(1,2) (SEQ ID NO:15) (SEQ ID (SEQ ID (SEQ ID NO:22) (SEQ IDNO:24) (SEQ ID NO:27) (SEQ ID NO:18) NO:20) NO:30) FAOT IIGSGAGAGVVAAGSTFGGG NWSACLKTP CGFCHLGC IGKNLTLHPVS SAHQMSTCRMSG PTASGANPM (SEQ IDNO:16) (SEQ ID (SEQ ID (SEQ ID NO:23) (SEQ ID NO:25) (SEQ ID NO:28) (SEQID NO:18) NO:20) NO:31) FAO1 IIGSGAGAGVVA AGSTFGGG NWSACLKTP CGFCYLGCIGKNLTLHPVS SAHQMSSCRMSG PTASGANPM (SEQ ID NO:16) (SEQ ID (SEQ ID (SEQID NO:13) (SEQ ID NO:25) (SEQ ID NO:29) (SEQ ID NO:18) NO:20) NO:31)FAO2a IIGSGAGAGVMA AGSTLGGG NWSACLKTP CGFCYLGC IGKNLTLHPVS SAHQMSTCRMSGPTASGANPM (SEQ ID NO:14) (SEQ ID (SEQ ID (SEQ ID NO:13) (SEQ ID NO:25)(SEQ ID NO:28) (SEQ ID NO:19) NO:20) NO:31) FAO2b IIGSGAGAGVMA AGSTLGGGNWSACLKTP CGFCYLGC IGKNLTLHPVS SAHQMSTCRMSG PTASGANPM (SEQ ID NO:14)(SEQ ID (SEQ ID (SEQ ID NO:13) (SEQ ID NO:25) (SEQ ID NO:28) (SEQ IDNO:19) NO:20) NO:31) FAOCA IIGSGAGSGVVA AGSTFGGG NWSACIKTP CGFCHLGCIGANLTLHPVT SAHQMSSCRMSG PTASGANPM (SEQ ID NO:17) (SEQ ID (SEQ ID (SEQID NO:23) (SEQ ID NO:26) (SEQ ID NO:29) (SEQ ID NO:18) NO:21) NO:31)¹Amino acids in bold indicate amino acid differences from signaturepeptide ²(X) indicates that any amino acid may be at this position

REFERENCES

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1. An isolated nucleic acid molecule that encodes a fatty alcoholoxidase having the amino acid sequence of SEQ ID NO:2.
 2. The isolatednucleic acid molecule of claim 1 comprising the nucleotide sequence ofSEQ ID NO:1.
 3. A vector comprising the isolated nucleic acid moleculeof claim
 1. 4. A vector comprising the isolated nucleic acid molecule ofclaim
 2. 5. The vector of claim 3 wherein the vector is a plasmid,phagemid, phage, cosmid, or linear DNA vector.
 6. The vector of claim 4wherein the vector is a plasmid, phagemid, phage, cosmid, of linear DNAvector.
 7. A host cell transformed with the vector of claim
 3. 8. A hostcell transformed with the vector of claim
 4. 9. The host cell of claim 7wherein the cell is a bacterial cell, fungal cell, insect cell; animalcell or plant cell.
 10. The host cell of claim 8 wherein the cell is abacterial cell, fungal cell, insect cell, animal cell or plant cell. 11.The fungal cell of claim 9 wherein the fungal cell is a yeast cellselected from the group consisting of Yarrowia sp., Bebaromyces sp.,Saccharomyces sp., Schizosaccharomyces sp., Pichia sp. and Candida sp.12. The fungal cell of claim 10 wherein the fungal cell is a yeast cellselected from the: group consisting of Yarrowia sp., Bebaromyces sp.,Saccharomyces sp., Schizosaccharomyces sp., Pichia sp. and Candida sp.13. The isolated nucleic acid molecule of claim 1 further comprising anisolated nucleic acid molecule encoding the signature motif of SEQ IDNO:13, where the encoded fatty alcohol oxidase continues to have fattyalcohol oxidase activity.
 14. The isolated molecule of claim 13, whereinSEQ ID NO: 13 is encoded by the nucleotide sequence: TGY GGN TTY TGY TAYYTN GGN TGY of SEQ liD NO. 32, wherein: Y is C or T, and N is A or G, orC or T.
 15. An isolated nucleic acid molecule comprising a nucleotidesequence which encodes the signature motif of SEQ ID NO:13 and whichhybridizes under high stringency conditions to nucleotides 1941–4054 ofthe nucleotide sequence of SEQ ID NO:1, wherein the nucleic acidmolecule encodes a fatty alcohol oxidase and said high stringencycondition is 0.1–1×SSC/0.1% (w/v) SDS at greater than or equal to 60° C.for 1–3 hours.
 16. An isolated nucleic acid molecule comprising an openreading frame (ORF) for a fatty alcohol oxidase (FAO) polynucleotidefrom Candida tropicalis wherein the ORF is operably linked to a promoterwhich regulates the expression of the ORF and said ORF encodes the aminoacid sequence of SEQ. ID NO:2.
 17. The isolated nucleic acid molecule ofclaim 16 wherein the ORF comprises nucleotides 1941–4054 of thenucleotide sequence SEQ ID NO:1.
 18. The isolated nucleic acid moleculeof claim 16 wherein the promoter is a cytochrome p450 monooxygenasepromoter.
 19. The isolated nucleic acid molecule of claim 18, whereinthe promoter comprises the nucleotide sequence of SEQ ID NO:7.
 20. Avector comprising the isolated nucleic acid molecule of claim
 16. 21. Avector comprising the isolated nucleic acid molecule of claim
 17. 22. Avector comprising the isolated nucleic acid molecule of claim
 18. 23. Avector comprising the isolated nucleic acid molecule of claim
 19. 24.The vector of claim 20 wherein the vector is a plasmid, phagemid, phage,cosmid, or linear DNA vector.
 25. The vector of claim 21 wherein thevector is a plasmid, phagemid, phage, cosmid, or linear DNA vector. 26.The vector of claim 22 wherein the vector is a plasmid, phagemid, phage,cosmid, or linear DNA vector.
 27. The vector of claim 23 wherein thevector is a plasmid, phagemid, phage, cosmid, or linear DNA vector. 28.An isolated host cell transformed with the isolated nucleic acidmolecule of claim
 16. 29. An isolated host cell transformed with theisolated nucleic acid molecule of claim
 17. 30. An isolated host celltransformed with the isolated nucleic acid molecule of claim
 18. 31. Anisolated host cell transformed with the isolated nucleic acid moleculeof claim
 19. 32. An isolated host cell transformed with the vector ofclaim
 20. 33. An isolated host cell transformed with the vector of claim21.
 34. An isolated host cell transformed with the vector of claim 22.35. An isolated host cell transformed with the vector of claim
 23. 36. Amethod of producing a fatty alcohol oxidase (FAO1) protein, said methodcomprising: transforming a suitable isolated host cell with a DNAsequence encoding a protein having the amino acid sequence of SEQ IDNO:2; and culturing the transformed cell and expressing the fattyalcohol oxidase (FAO1) protein.